CN107872982B

CN107872982B - Systems and methods for improved tissue sensing-based electroporation

Info

Publication number: CN107872982B
Application number: CN201680026625.2A
Authority: CN
Inventors: A·巴哈拉米; A·E·丹尼森; C·S·海登; R·J·康诺利; R·H·皮尔斯; D·W·布朗; E·T·约翰逊; R·R·拉格兰; J·坎贝尔
Original assignee: OncoSec Medical Inc
Current assignee: Hongnian Development Co ltd
Priority date: 2015-03-31
Filing date: 2016-03-31
Publication date: 2022-10-28
Anticipated expiration: 2036-03-31
Also published as: JP2018510015A; WO2016161201A3; ES2807439T3; US20230001189A1; EP3695877A1; JP2021094467A; PL3277368T3; CN115737104A; US20190117964A1; JP6860497B2; EP3277368A2; JP2023029619A; EP3277368B1; HK1250675A1; CN107872982A; CA2981474A1; WO2016161201A2; US11318305B2; DK3277368T3

Abstract

The present disclosure relates to an adaptive control method for controlling an Electroporation (EP) pulse parameter during EP of a cell or tissue using an EP system, comprising: a system is provided for adaptive control to optimize EP pulse parameters including EP pulse parameters, applying a voltage and a current excitation signal to a cell, obtaining data from current and voltage measurements, and processing the data to separate desirable data from undesirable data, extracting a relevant feature from the desirable data, applying at least a portion of the relevant feature to a trained diagnostic model, estimating EP pulse parameters based on results of the applied relevant feature, wherein an initialized EP pulse parameter is based on the trained model and the relevant feature, thereby optimizing the EP pulse parameters, and applying a first EP pulse by a generator based on the first pulse parameters.

Description

Systems and methods for improved tissue sensing-based electroporation

Reference to related applications

The present application claims that U.S. provisional patent application No. 62/214,807 filed on 9/4 th of 2015 entitled "system and METHOD FOR optimizing ELECTROPORATION (SYSTEM AND METHOD FOR OPTIMIZED ELECTROPORATION)" and priority of U.S. provisional patent application No. 62/214,872 filed on 9/4 th of 2015 entitled "system and METHOD FOR OPTIMIZED CATHETER-BASED ELECTROPORATION (SYSTEM AND METHOD FOR OPTIMIZED CATHETER ELECTROPORATION-BASED ELECTROPORATION"), each of which is filed on 3/31 th of 2015 entitled "FOCUSED PULSE ADDITION ELECTROPORATION (FOCUSED PULSE ADDITION ELECTROPORATION)", U.S. provisional patent application No. 62/141,142 filed on 3 rd of 2015 entitled "ELECTROCHEMICAL TISSUE SENSING (DEVICE FOR ELECTROPORATION"), U.S. provisional patent application No. 62/141,142 filed on 3 rd of 2015 3 th of 2015 entitled "DEVICE FOR ELECTROCHEMICAL TISSUE SENSING" (filed on 3 th of, "and patent application No. 62/141/141,164 filed on 3 th of 2015 filed on 3 th of" FOR IMPROVED delivery of THERAPEUTIC agent FOR use by "DEVICE FOR the same as disclosed IN provisional patent application No. 62/214/141, 3532, filed on 3 th of" filed on 3 th of 2015 3 th of this application, 3 th of provisional patent application No. (FOR IMPROVED FOR delivery of therapy) (FOR use).

Technical Field

The present invention relates generally to the use of control systems to improve the electroporation process and increase the permeability of cells, and more specifically to methods and apparatus for optimized application of controlled electric fields for delivery of therapeutic moieties into cells by electroporation therapy (EPT), also known as Cell Poration Therapy (CPT) and Electrochemotherapy (ECT).

Background

In the 1970 s it was found that pores could be created in cells using electric fields without permanent damage. This finding makes it possible to insert macromolecules into the cytoplasm of cells. It is well known that therapeutic moieties such as pharmacological compounds can be incorporated into living cells by a process known as electroporation. Genes or other molecules are injected into living cells and a short pulse of high electric field is applied. The cell membrane becomes briefly porous and a gene or molecule enters the cell where it can modify the genome of the cell.

In the treatment of certain types of cancer with chemotherapy, it is necessary to use a sufficiently high dose of the drug to kill cancer cells without killing an unacceptably high number of normal cells. This can be achieved if the chemotherapeutic drug can be inserted directly inside the cancer cells. Some anti-cancer drugs, such as bleomycin (bleomycin), generally do not penetrate the membrane of certain cancer cells effectively. However, electroporation makes it possible to insert bleomycin into cells.

Treatment is typically carried out by injecting an anti-cancer drug directly into the tumor and applying an electric field to the tumor between a pair of electrodes. The field strength must be adjusted exactly and reasonably so that electroporation of the cells of the tumor takes place without damage or at least with minimal damage to any normal or healthy cells. This can be easily done for external tumours, typically by applying electrodes to opposite sides of the tumour such that the electric field is between the electrodes. When the field is uniform, the distance between the electrodes can then be measured, and a suitable voltage according to the formula E = V/d can then be applied to the electrodes (E = field strength in V/cm; V = voltage in volts; and d = distance in cm). When a large or internal tumor is to be treated, it is not easy to properly position the electrodes and measure the distance between the electrodes.

Treatment of a subject using cell poration therapy provides a means to avoid the deleterious effects typically associated with administration of anti-cancer or cytotoxic agents. This treatment would allow the introduction of these agents to selectively damage or kill undesirable cells while avoiding surrounding healthy cells or tissue. However, one problem with the use of electroporation techniques is that diseased tissue, especially cancerous tissue, may be quite heterogeneous, requiring adjustment of electroporation conditions. Accordingly, the present invention provides for the use of electrochemical impedance spectroscopy methods in combination with adaptive control methods for EP to maximize electroporation of desired tissue while minimizing tissue damage.

Disclosure of Invention

Therefore, there is a need to implement a control system that uses tissue sensing based feedback to optimize the EP process with tumor specific measurements taken before and between each EP pulse.

According to some embodiments, a system for providing adaptive control to optimize EP pulse parameters during EP of cells and tissues using an Electroporation (EP) device includes a measurement device, an initialization module, a generator, a controller, and a memory module. The measurement device is configured to measure dielectric and conductive properties of cells and tissues, and comprises: a voltage sensor to measure a voltage across the tissue resulting from each of the excitation signal and the EP pulse applied to the tissue; and a current sensor to measure a current across the tissue resulting from the excitation signal and the at least one applied EP pulse. The initialization module is configured to initialize EP pulse parameters for performing electroporation in a cell or tissue, wherein the initialized EP pulse parameters are based at least in part on the at least one trained model. The generator is configured to apply at least one of an excitation signal and an EP pulse to tissue. The voltage sensor and the current sensor of the measurement device measure voltage and current across cells of the tissue in response to application of the excitation signal. The controller is configured to receive signals from the measurement device corresponding to at least one of the excitation signal and the EP pulse in relation to the measured sensor data, to fit the data to at least one trained model and to process the data into diagnostics and updated control parameters. The controller includes: a pre-processing module to receive signals relating to data from current and voltage measurements and process the data to separate desirable data from undesirable data; a feature extraction module to extract relevant features from the consensus data; a diagnostic module to apply at least a portion of the relevant features of the desired data to at least one trained diagnostic model; and a pulse parameter estimation module to estimate at least one of the initialized pulse parameter and the subsequent pulse parameter based on results of at least one of the measured data, the diagnostic module, and the feature extraction module. The memory module stores desirable and undesirable data, sensor data, and trained models for feature extraction by the controller.

In some embodiments, the EP device includes a central probe, an applicator, and at least two oppositely charged electroporation electrodes (EPEs). A central probe defines at least a central lumen and extends from a proximal end to a distal end, at least a portion of the central probe having a helical geometry to create a channel for delivery of the treatment portion to tissue. The portion of the central probe has at least one injection port positioned along a helical geometry. The proximal end of the central probe is configured to receive a treatment portion delivered to the central probe, and the distal end of the central probe is open to define an opening for delivery of the treatment portion to tissue and has a shape configured to pierce the tissue. The applicator at least partially houses the central probe and has a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator. The at least two oppositely charged EPEs are configured to be positioned surrounding tissue and adapted to extend from a proximal end to a distal end. The distal end has a needle shape configured to pierce tissue. The measurement device is coupled to the EPE, and the EPE is adapted to be coupled to a generator to receive at least one of an excitation signal and an electrical waveform for the EP pulse.

In some embodiments, the EP device includes a central probe, at least one lane line, a ramp, an electrical connector, a small aperture connector, a handle, and at least two oppositely charged electrodes. The central probe defines at least a central lumen and has a proximal end and a closed distal end. The tip of the distal end has a needle shape configured to pierce tissue and has at least one exit port positioned at a predetermined location from the distal end. An exit port fluidly connects the central lumen to an exterior of the central probe. The at least one access line is positioned in the central lumen and is slidable within the central probe, and has a proximal end positioned in the central probe and a distal end configured to extend outside of the central probe and retract into the central lumen through the exit port. The distal end of the access wire has a tip configured to pierce through tissue and define an opening through which at least a portion of the access wire enters the tissue to create a fluid channel through which the therapeutic portion is delivered to the tissue. The treatment portion is delivered from the central lumen into the channel through the exit port. The ramp is integrally formed or coupled with an inner surface of the central probe, the inner surface defining a central lumen, and the ramp is configured to contact and guide the access line to exit the central probe outside of the central probe. An electrical connector electrically connects the central probe and the access line to the generator. A small aperture connector is connected to the central probe for delivery of the treatment portion. A handle houses, at least in part, the electrical connector and is coupled to the central probe and the proximal end of the access line to facilitate a penetration depth of the central probe and the distal end of the access line. The at least two oppositely charged electrodes are configured to be positioned surrounding tissue and extend from a proximal end to a distal end. The distal tip has a needle shape configured to pierce tissue. The electrode is adapted to be coupled to a generator, receive at least one electrical waveform from the generator, and supply at least one excitation signal and at least one EP pulse to tissue. A measurement device is coupled to the electrode.

In some embodiments, the EP device includes a trocar comprising a cannula and an obturator, at least two oppositely charged electrodes, and a central probe. The cannula extends from a proximal end to an open distal end and defines a first lumen configured to receive an obturator. The obturator extends from the proximal end to the distal end. The distal end has a sharpened shape configured to pierce through the skin, penetrate into a body cavity, and form a path through which the cannula may be at least partially inserted into the cavity. The obturator is configured to slide within the first lumen, and a distal end of the obturator is configured to extend outside of the first lumen through the open distal end of the cannula. The at least two oppositely charged electrodes are retractably disposed at a distal end of the anchor and are configured to be positioned around tissue. A measurement device is coupled to the electrode and the electrode is adapted to be coupled to a generator, receive at least one electrical waveform from the generator, and supply at least one excitation signal and EP pulse to the zone. A central probe is retractably disposed at the distal end of the anchor and has an inner surface defining a central lumen and extending from the distal end of the anchor. At least a portion of the central probe has a helical geometry configured to create a channel for delivery of the treatment portion to the tissue. The distal end of the central probe has a shape configured to pierce tissue and is open to define an opening for delivery of the treatment portion to the tissue.

In some embodiments, the EP device comprises an electroporation cuvette housing comprising an array of electroporation electrodes (EPEs), an array of Electrical Measurement Electrodes (EMEs), wherein the EPEs and EMEs are offset; and a rod delivery system comprising at least one injection probe defining a first lumen. The injection probe extends from a proximal end to a distal end thereof and has an elongated cylindrical shape. The distal end of the injection probe has a needle shape and is open for delivery of the therapeutic moiety to the cells. The generator is configured to supply EP pulses at a plurality of waveforms to the array of EPEs and is configured to supply excitation signals at a plurality of waveforms to the array of EMEs. The EP device further includes an electrical connector electrically connecting the array of EPEs and EMEs to the generator, and a switching mechanism between the electrical connector and the generator.

In some embodiments, both the EPEs and EMEs are configured as EPEs, i.e., the electrodes are all EPEs that are capable of switching between EP and Electrochemical Impedance Spectroscopy (EIS) modes. The generator is configured to supply the EPE with EP pulses at the plurality of waveforms in the EP mode and with excitation signals at the plurality of waveforms in the EIS mode, the measurement device is coupled to the EPE, and the switching mechanism is adapted to switch the generator between the EIS and EP modes.

According to some embodiments, an adaptive control method for controlling an Electroporation (EP) pulse parameter during EP of a cell or tissue using an EP system, comprises: a) providing any of the EP devices described herein, b) initializing EP pulse parameters for performing EP in the cells or tissue by an initialization module, the initialized EP pulse parameters being based at least in part on at least one trained model, c) applying voltage and current excitation signals to the cells and tissue by the generator and measuring voltage and current across the cells and tissue corresponding to the applied excitation signals by the measurement device, d) obtaining data from the current and voltage measurements by the controller and processing the data to separate desired data from undesired data, e) extracting relevant features from the desired data by the controller, f) applying at least a portion of the relevant features of the desired data to at least one trained diagnostic model by the controller, g) estimating, by the controller, the EP pulse parameters based on the results of the applied relevant features to the trained model, wherein the initialized EP pulse parameters are based on the at least one trained model and the relevant features, thereby optimizing the EP pulse parameters, and h) applying, by the generator, a first EP pulse based on the first pulse parameters.

In some embodiments, the adaptive control method further comprises predicting, by the controller, subsequent EP pulse parameters after the first EP pulse has been applied using a trained model based on previous EP pulses and a change in at least one of the correlation characteristics between the applied EP pulses.

In some embodiments, the adaptive control method further comprises generating, by the controller, a diagnostic response based at least in part on the applying. The diagnostic response includes a) tissue detection, b) tumor type detection, c) needle placement detection, d) co-localization detection, and e) cell infiltration detection.

In some embodiments, the adaptive control method further comprises: f) Applying, by a generator, a subsequent EP pulse based on subsequent EP pulse parameters, and g) repeating the applying voltage and current excitation signals, repeating the measuring cell or tissue, repeating the obtaining data and separating desirable data from undesirable data; repeating said extracting relevant features; and repeating the applying until i) a predetermined limit of the number of EP pulse sequences or cycles of EP pulses is reached, or ii) a diagnostic decision of the adaptive control method is terminated in response to a prompt.

In some embodiments, the adaptive control method further comprises storing the desired data in a memory module.

In some embodiments, the at least one trained model is trained using empirical data observed during initial operation of the EP system using fixed EP pulse parameters.

In some embodiments, the adaptive control method further comprises determining the dielectric and conductive properties of cells and tissues resulting from the applied excitation signal.

In some embodiments, the dielectric and conductive properties are determined by applying a band-limited signal that repeats over a fixed frequency range.

In some embodiments, the adaptive control method further comprises validating current and voltage sensors of the measurement device, whereby measured data is obtained to assess the quality of the data, and the validating comprises statistically analyzing the quality of the measured data.

In some embodiments, separating the desirable data from the undesirable data includes at least one of: a) de-noising the sensor signal, b) removing Direct Current (DC) bias from the sensor signal, c) scaling the data based on a normalized value, wherein the normalized value comprises a standard deviation, d) mean filtering, and e) removing outliers from the data.

In some embodiments, the characteristic is derived from a parametric model fit of magnitude and phase measurements of the voltage and current signals selected from the group consisting of intracellular resistance, extracellular resistance, solution resistance, thin film capacitance, admittance, constant phase element index, and charge time constant.

In some embodiments, a parametric model fit of the magnitude and phase measurements of the voltage and current signals of the excitation voltage and current signals applied to the cells and tissues is determined by cross-correlating the excitation voltage and current signals with known reference signals stored in a memory module.

In some embodiments, the dielectric and conductive properties of the cells or tissue are determined by the magnitude ratio and phase difference of the excitation voltage and current applied to the cells or tissue.

In some embodiments, the characteristic is derived from a magnitude ratio or phase difference of the excitation voltage and current signals. The features include: a) A magnitude ratio and a phase difference value of the excitation voltage and current signals at a fixed frequency, b) at least one of an average, a median, a maximum, and a minimum of: i) Magnitude ratio or phase difference of the excitation voltage and current signal magnitudes over a narrow frequency band, and ii) magnitude ratio or phase difference of the excitation voltage and current signal magnitude phases over a wide frequency band, and c) curvature, slope, and noise of the magnitude ratio or phase difference of the excitation voltage and current signals with respect to frequency.

According to some embodiments, a system for Electroporation (EP) of cells in tissue of a subject comprises: a) An electroporation rod housing comprising i) an array of electroporation electrodes (EPEs); and ii) an array of Electrochemical Impedance Spectroscopy (EIS) electrodes (EISE), wherein the EPE and EISE are offset, b) an EP power supply configured to supply electrical signals at a plurality of waveforms to the array of EPEs, c) an EIS power supply configured to supply electrical signals at a plurality of waveforms to the array of EISE, d) an electrical connector electrically connecting the array of EPEs to the EP power supply, and e) an electrical connector electrically connecting the array of EISEs to the EIS power supply, and f) an EIS sensor.

According to some embodiments, the system further comprises a rod delivery system configured to deliver the therapeutic moiety to the cell, the delivery system comprising at least one injection probe defining a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic moiety to the cell.

According to some embodiments, a system for Electroporation (EP) of cells in tissue of a subject comprises: a) an electroporation rod housing comprising an array of electrodes, b) an EP power supply configured to supply electrical signals at a plurality of waveforms to the array of electrodes, c) an EIS power supply configured to supply electrical signals at a plurality of waveforms to the array of electrodes, d) an electrical connector electrically connecting the array of electrodes to the EP power supply, e) an electrical connector electrically connecting the array of electrodes to the EIS power supply, f) a switching mechanism between the electrical connector and the power supply, and g) an EIS sensor.

In some embodiments, the system thus further comprises a rod delivery system configured to deliver the therapeutic moiety to the cell, the delivery system comprising at least one injection probe defining a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongate cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic moiety to the cell.

In some embodiments, the electrode is a needle configured to penetrate the skin and contact cells in the electric field region.

In some embodiments, the electrode is a non-penetrating contact.

According to some embodiments, a method for electroporating cells of a tissue in a patient comprises: a) providing any of the EP systems described herein, b) inserting electrodes into tissue, c) applying at least one voltage pulse to the EIS electrodes from the EIS power supply to determine tissue parameters, d) calculating the voltage pulse to be used for electroporation using an electronic signal processing device, and e) applying at least one voltage pulse between pairs of electrodes in the EP electrode array inserted into the tissue so as to establish an electric field in cells of the tissue sufficient to cause electroporation of cells in the tissue.

In some embodiments, the method further comprises: a) Providing a rod delivery system configured to deliver a Therapeutic Moiety (TM) to a cell, the delivery system comprising at least one injection defining a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic moiety to the cell; and b) delivering the TM to the cell.

In some embodiments, the TM is delivered prior to, simultaneously with, or after electroporation.

In some embodiments, the TM is locally injected into the tissue.

In some embodiments, the method is in vivo.

In some embodiments, the TM is a nucleic acid.

In some embodiments, the cell is a tumor cell.

In some embodiments, the cell is a melanoma or basal cell carcinoma cell.

In some embodiments, the electric field ranges from approximately 10V/cm to about 2000V/cm.

In some embodiments, the number of electrical pulses applied ranges from 1 to 100.

In some embodiments, the duration of each electrical pulse ranges from about 10 μ s to about 100ms in duration.

In some embodiments, the at least one electrical pulse is selected from the group consisting of: square wave pulses, exponential wave pulses, unipolar vibration wave forms, and bipolar vibration wave forms.

In some embodiments, each electrical pulse comprises a square wave pulse.

According to some embodiments, a method of electroporating an agent into cells of a tissue comprises: a) introducing a therapeutic agent into tissue of a patient in need of treatment, b) performing tissue impedance sensing to determine a suitable EP protocol, c) using an electrode apparatus placed in contact with the tissue to deliver voltage pulses that establish an electric field sufficient to introduce the therapeutic agent into cells of the tissue by means of electroporation, wherein the electrode apparatus comprises i) a support member having disposed thereon two or more opposing pairs of needle electrodes arranged relative to each other to form an electrode array, and ii) a power supply in electrical communication with the pairs of needle electrodes disposed in the support member, wherein the power supply provides voltage pulses to at least two of the opposing pairs of needle electrodes to effect electroporation.

In some embodiments, an apparatus for delivering a therapeutic moiety to cells in a treatment region of a tissue comprises: a) A central probe defining at least a central lumen and extending from a proximal end to a distal end, at least a portion of the central probe having a helical geometry to create a channel for delivery of a treatment portion to tissue, the portion of the central probe having at least one ejection port positioned along the helical geometry. The proximal end of the central probe is open and fluidly connects the first central lumen with the lumen of an injector through which the therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce tissue. The means for delivering further comprises: b) An applicator at least partially housing a central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator.

In some embodiments, the device further comprises at least one electrode pair positioned on the portion of the central probe.

In some embodiments, the distal end of the central probe is closed.

In some embodiments, at least one of the diameter of the first lumen of the central probe, the outer diameter of the central probe, the helical diameter, and the spacing is adjustable to vary the distribution and volume of the delivered treatment portions.

In some embodiments, the central probe is actuated to advance toward and through the distal end of the central probe and through the tissue.

In some embodiments, the apparatus further comprises: a) An electrical connector that electrically connects the central probe to a source of electrical power, and b) a handle that houses the electrical connector and is coupled to the applicator.

In some embodiments, the proximal end of the central probe is formed of or coated with a non-conductive material to prevent or reduce the generation of an electric field at the portion.

In some embodiments, the device further comprises an electroporation system comprising at least two oppositely charged electroporation electrodes configured to be positioned around the zone, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce tissue. The electrode is adapted to be coupled to an electrode power supply, receive at least one electrical waveform from the power supply, and supply a pulsed electric field sufficient for electroporation to the region.

In some embodiments, the electrodes are housed at least partially in the applicator, positioned around the central probe and configured to be deployed from the applicator to surround the zone.

In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate extension and retraction of the central probe, and to actuate extension and retraction of the electroporation electrodes.

In some embodiments, the device further comprises a sensor system configured to sense the capacitance of the cell membrane. The sensor system includes: a) a pair of capacitance or EIS sensing electrodes powered by a low voltage power supply, b) a voltage sensor configured to sense a voltage or voltage drop across the cell membrane, c) a current sensor configured to sense a current across the cell membrane, and d) an electronic signal processing device configured to process the voltage drop and current across the cell membrane and determine the capacitance of the cell membrane.

In some embodiments, the central probe is an electrode probe connected to an electrode power supply configured to generate an electric field between the central probe and the electroporation electrode to facilitate electroporation.

In some embodiments, the device further comprises at least a second probe having a helical geometry defining at least a second lumen and extending from a proximal end to a distal end of the other probe, at least a portion of the other probe having at least a second channel configured to generate delivery of the treatment portion to the tissue. The proximal end of the other probe is open and fluidly connects the second lumen with a lumen of an injector through which therapeutic agent is delivered to the other probe. The distal end of the other probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce through tissue. The other probe is housed in the applicator and the portion of the other probe is configured to extend outside of the applicator to contact tissue and retract into the applicator.

According to some embodiments, an apparatus for delivering a treatment portion to cells in a treatment region of a tissue comprises: a) A central probe defining at least a first lumen and extending from a proximal end to a distal end, at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the treatment portion to the tissue. The portion of the central probe is formed from or coated with an electrically conductive material. The proximal end of the central probe is open and fluidly connects the first lumen with the lumen of an injector through which the therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce tissue. The device further comprises: b) An applicator housing a central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator, and c) at least one distal electrode positioned at the distal end of the applicator and configured to generate an electric field with the portion of the central probe.

In some embodiments, the at least one distal electrode is configured based on a loop configuration, a straight wire configuration, a helical wire configuration, or a collapsible loop configuration.

In some embodiments, the device further comprises at least one injection port positioned on the portion of the central probe.

In some embodiments, the distal electrode is configured to be positioned outside of tissue.

In some embodiments, the distal electrode is configured to be positioned below a surface of tissue.

In some embodiments, the distal electrode is formed from a helical wire configuration, positioned below the surface of the tissue, and the helices of the central probe and the distal electrode are wound in opposite directions.

In some embodiments, the electrodes are housed in an applicator, positioned around the central probe and configured to be deployed from the applicator to surround the zone.

In some embodiments, the device further comprises a sensor system configured to sense a capacitance of the cell membrane. The sensor system includes: a) a pair of capacitive sensing or EIS electrodes powered by a low voltage power supply, b) a voltage sensor configured to sense a voltage or voltage drop across the cell membrane, c) a current sensor configured to sense a current across the cell membrane, and d) an electronic signal processing device configured to process the voltage drop and current across the cell membrane and determine the capacitance of the cell membrane.

According to some embodiments, an apparatus for delivering a therapeutic moiety to cells in a treatment zone of a tissue comprises: a) A central probe having an inner surface defining at least a first central lumen and extending from a proximal end to a distal end of the central probe, at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in tissue and to create a channel for delivery of the treatment portion to the tissue, wherein the portion of the central probe is formed of or coated with an electrically conductive material. The proximal end of the central probe is open and fluidly connects the central lumen with the lumen of the injector through which the therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce tissue. The apparatus further comprises: b) An applicator housing a central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator, c) at least one straight probe having an open proximal end and a distal end for delivery of the treatment portion to the tissue, and a vertical axis coaxially aligned with a central axis of a diameter of the central probe and configured to generate an electric field with the portion of the central probe.

In some embodiments, the helical probe is configured to transmit acoustic energy received from an acoustic horn mounted to the distal end of the applicator.

In some embodiments, the device further comprises a sensor system configured to sense a capacitance of a cell membrane, the sensor system comprising:

according to some embodiments, a method for delivering a treatment portion to a treatment area of a tissue includes a) providing a device for delivering the treatment portion to the treatment area of the tissue. The apparatus comprises i) a central probe and ii) an applicator. A central probe has at least a first central lumen and extends from a proximal end to a distal end, at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the treatment portion to the tissue. A portion of the central probe has a plurality of injection ports positioned along a spiral geometry. The proximal end of the central probe is open and fluidly connects the central lumen with the lumen of an injector through which the therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce through tissue. The applicator houses a central probe and has a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator. The method further comprises: b) Contacting the central probe to diseased cells in a treatment area of the tissue, c) actuating and extending the central probe in an axial direction from the applicator, d) piercing the tissue with at least a portion of the central probe and creating an opening through which at least a portion of the central probe enters the tissue to create a fluid channel for delivery of the treatment portion to the tissue, and e) injecting the treatment portion into the first central lumen and delivering the treatment portion to the tissue through the at least one jet port and the open distal end of the central probe.

In some embodiments, the method further comprises f) providing an electroporation system comprising at least two oppositely charged electroporation electrodes configured to be positioned around the zone. The electroporation electrode is adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce tissue, and the electroporation electrode is adapted to be coupled to a source of electrical power. The method further includes g) contacting a region of tissue with the electroporation electrode, h) delivering an electrical pulse from the electrical power source to the electrode, and i) applying a pulsed electric field sufficient for electroporation to the region from the electroporation electrode.

In some embodiments, the method further comprises providing a sensor system that senses the capacitance of the cell membrane. The capacitive sensing includes: a) contacting the tissue with at least one pair of capacitive sensing electrodes powered by a low voltage power supply, b) transmitting a low power interrogation signal through the low voltage power supply to the at least one pair of capacitive sensing electrodes to generate a low intensity electric field excitation in the region, c) sensing the voltage or voltage drop across the cell membrane by a voltage sensor, d) sensing the current across the cell membrane by a current sensor; and e) determining, by the electronic signal processing means, the capacitance of the cell membrane based on the voltage drop and the current across the cell membrane.

According to some embodiments, a method for delivering a treatment portion to a treatment area of a tissue includes a) providing a device for delivering the treatment portion to the treatment area of the tissue. The device includes i) a central probe connected to a source of electrical power and having an inner surface defining at least a first central lumen and extending from a proximal end to a distal end of the central probe. At least a portion of the central probe has a helical geometry configured to enhance anchoring of the central probe in the tissue and to create a channel for delivery of the treatment portion to the tissue. The portion of the central probe is formed from or coated with an electrically conductive material. The proximal end of the central probe is open and fluidly connects the central lumen with the lumen of an injector through which the therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce tissue. The apparatus further comprises: ii) an applicator containing a central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact tissue and retract into the applicator, and iii) at least one distal electrode positioned at the distal end of the applicator, connected to a source of electrical power and configured to generate an electric field with the portion of the central probe. The method further includes b) contacting the central probe and the distal electrode to diseased cells in a treatment area of the tissue, c) actuating and extending the central probe and the distal electrode in an axial direction from the applicator, d) piercing the tissue with the distal electrode and with at least a portion of the central probe and creating an opening through which at least a portion of the central probe enters the tissue to create a fluid channel for delivery of the treatment portion to the tissue, e) injecting the treatment portion into the first central lumen and delivering the treatment portion to the tissue through the at least one ejection port and the open distal end of the central probe, f) delivering electrical pulses from the electrical power source to the distal electrode and the central probe, g) applying a pulsed electrical field sufficient for electroporation to the area from the distal electrode and the central probe, and h) retracting the distal electrode and the central probe from the tissue.

According to some embodiments, an apparatus for delivering a therapeutic moiety to a region of target cells of a tissue comprises: a) A central probe defining at least a first lumen and having a proximal end and a closed distal end, a tip of the distal end having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the first lumen to an exterior of the central probe, and b) at least one access line positioned in the first lumen and slidable within the central probe, the access line having a proximal end positioned in the central probe and a distal end configured to extend to the exterior of the central probe and retract into the first lumen through the exit port, the tip of the distal end of the access line having a shape configured to pierce through tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which a therapeutic portion is delivered to the tissue. The treatment portion is delivered from the first lumen into the channel through the exit port. The apparatus further comprises: c) A ramp integrally formed or coupled with the first lumen, the ramp configured to contact and guide the access line to exit the central probe outside of the central probe, d) an electrical connector that electrically connects the central probe and the access line to a power source, e) a small aperture connector that connects the central probe to a syringe for delivery of the treatment portion, and f) a handle that at least partially houses the electrical connector and is coupled to a proximal end of the central probe and the access line to facilitate a penetration depth of a distal end of the central probe and the access line.

In some embodiments, the device further comprises an electroporation system comprising at least two oppositely charged electrodes configured to be positioned surrounding the region of target cells, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to the source of electrical power, receive an electrical waveform from a power supply, and supply a pulsed electrical field sufficient for electroporation to the region of target cells.

In some embodiments, the electrode surrounds the central probe.

In some embodiments, the device comprises a plurality of exit ports and the plurality of access lines are configured to simultaneously extend outside of the central probe and configured to retract into the central lumen of the central probe through the exit ports.

In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate extension and retraction of the access line, and to actuate extension and retraction of the electrode.

In some embodiments, the device further comprises a catheter shaft that surrounds an outer surface of the central probe to support and protect the central probe during insertion into the body with tissue.

In some embodiments, the access line includes a cutting blade at a tip located at a distal end of the access line.

In some embodiments, the cutting blade at the distal end is configured to enter tissue and is configured to rotate about a central axis of the cutting blade to form a fluid channel.

In some embodiments, the angle at which the ramp contacts the lane line is adjustable to change the angle of the trajectory at which the lane line exits the central lumen.

According to some embodiments, an apparatus for delivering a therapeutic moiety to a region of target cells of a tissue comprises: a) A central probe defining at least a first lumen and having a proximal end and an open distal end, a tip of the distal end having a needle shape configured to pierce tissue and the open distal end fluidly connecting the first lumen to an exterior of the central probe, b) at least one access line positioned in the first lumen and being slidable within the central probe and having a proximal end positioned in the central probe and a distal end configured to extend to the exterior of the central probe and retract into the central lumen through the distal end of the central probe, the access line comprising a superelastic material configured to be heat-set in a curve, wherein the access line is adapted to elastically straighten when positioned in the central probe and to bend in a curve when extending to the exterior of the central probe to form a channel extending to cells, the access line having an elongated cylindrical shape and a distal end further configured to pierce through tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which a therapeutic portion is delivered to the tissue. The treatment portion is delivered from the first lumen into the channel through the exit port. The apparatus further comprises: b) A ramp integrally formed or coupled with an inner surface of the central probe, the ramp configured to contact and guide the access line to exit the central probe to an exterior of the central probe, c) an electrical connector that electrically connects the central probe and the access line to a power source, d) a small aperture connector that connects the central probe to a syringe for delivery of the treatment portion, and e) a handle that at least partially houses the electrical connector and is coupled to a proximal end of the central probe and the access line to facilitate a depth of penetration of a distal end of the central probe and the access line.

In some embodiments, the superelastic material is any one or combination of materials selected from the group consisting of NiTi, cu-Al-Ni, fe-Mn-Si, niTi-Zr, cu-Zr, ni-Al, and Cu-based alloys.

In some embodiments, the device further comprises at least two oppositely charged electrodes configured to be positioned around a region of target cells for cell therapy, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to a source of electrical power, receive an electrical waveform from a power supply, and supply a pulsed electrical field sufficient for electroporation to the target tissue region.

According to some embodiments, an apparatus for delivering a therapeutic moiety to a region of target cells of a tissue comprises: a) An injection probe defining at least a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongated cylindrical shape, the distal end having a needle shape and being open for delivering a treatment portion to the zone, b) a central probe, coupled to the injection probe and having an inner surface defining at least a second lumen, the central probe having a proximal end and a closed distal end, the distal tip having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined distance between the distal and proximal ends of the central probe, the exit port fluidly connecting the second lumen to the exterior of the central probe, c) at least one access line, positioned in the second lumen and slidable within the central probe, an access line having a proximal end positioned in the central probe and a distal end configured to extend outside of the central probe and retract into the second lumen through an exit port, a tip of the distal end of the access line having a shape configured to pierce through tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which a therapeutic portion is injected into the region by the injection probe, d) a ramp, integrally formed with or coupled to an inner surface of the central probe, defining an inner surface of the second lumen, and the ramp is configured to contact and guide the access line to exit the central probe outside of the central probe, e) an electrical connector, which electrically connects the central probe and the access line to the power source, and f) a handle that at least partially houses the electrical connector and is coupled to the proximal end of the central probe and the proximal ends of the injection probe and the access line to facilitate a penetration depth of the injection probe and the distal end of the central probe.

In some embodiments, the device further comprises at least two oppositely charged electrodes configured to be positioned around a target tissue area for cell therapy, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to an electrical power source, receive an electrical waveform from the electrical power supply, and supply a pulsed electrical field sufficient for electroporation to the target tissue area.

In some embodiments, the angle at which the ramp contacts the lane line is adjustable to change the corresponding trajectory angle at which the lane line exits the second lumen.

According to some embodiments, a method for delivering a therapeutic moiety to a region of target cells in a tissue includes a) providing a device for delivering the therapeutic moiety to the region of target cells of the tissue. The device comprises: i) A central probe having an inner surface defining at least a first central lumen and having a proximal end and a closed distal end, a tip of the distal end having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the central lumen to an exterior of the central probe, ii) at least one access line positioned in the central lumen and slidable within the central probe, the access line having a proximal end positioned in the central probe and a distal end configured to extend to the exterior of the central probe and retract into the central lumen through the exit port. The distal end of the access wire has a tip configured to pierce through tissue and define an opening through which at least a portion of the access wire enters the tissue to create a fluid channel through which the therapeutic portion is delivered to the tissue. The treatment portion is delivered from the first central lumen into the channel through the exit port. The apparatus further comprises: iii) a ramp integrally formed or coupled with an inner surface of the central probe, the inner surface defining a central lumen, and the ramp being configured to contact and guide the retrieval line to exit the central probe outside of the central probe, iv) an electrical connector electrically connecting the central probe and the retrieval line to a power source, v) a small aperture connector connecting the central probe to a syringe for delivery of the treatment portion, vi) a handle housing the electrical connector and coupled to a proximal end of the central probe and the retrieval line to facilitate a penetration depth of a distal end of the central probe and the retrieval line. The method further comprises: b) Inserting a central probe into diseased cells in a region of target cells, c) actuating and extending an access line from a central lumen in an axial direction of the central probe, a tip of a distal end of the access line having a needle shape that pierces through tissue and forms an opening through which at least a portion of the access line enters the tissue and creates a fluid channel through which a therapeutic portion is delivered, d) actuating a ramp integrally formed with or coupled to an inner surface of the central probe, the ramp contacting the access line and directing a trajectory of the access line through an exit port toward a distal end of the central probe, the exit port fluidly connecting the central lumen with an exterior of the central probe, e) piercing the tissue with the access line and creating an opening through which at least a portion of the access line enters the tissue to create a fluid channel for delivery of the therapeutic portion to the tissue, f) retracting the access line into the central lumen, and g) injecting the therapeutic portion into the central lumen and delivering the therapeutic portion to the tissue through the fluid channel.

In some embodiments, the method further comprises: a) The device is rotated for delivery at least once and the tissue is pierced with the access line to create an additional fluid channel for delivery of the treatment portion to the tissue, then the treatment portion is injected through the fluid channel and delivered to the tissue.

In some embodiments, the method further comprises: a) providing an electroporation system comprising at least two oppositely charged electroporation electrodes positioned configured to surround a region of a target cell, wherein the electroporation electrodes are adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce tissue, and the electroporation electrodes are adapted to be coupled to a power source, b) contacting the region of the target cell with the electroporation electrodes, c) delivering electrical pulses from the power source to the electrodes, and d) applying a pulsed electric field from the electroporation electrodes sufficient for electroporation to the region of the target cell.

According to some embodiments, a method for delivering a therapeutic moiety to a region of target cells in a tissue includes a) providing a device for delivering the therapeutic moiety to the region of target cells of the tissue. The device comprises: a) An injection probe defining at least a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongated cylindrical shape, the distal end having a needle shape and being open for delivering a treatment portion to the zone, b) a central probe, coupled to the injection probe and having an inner surface defining at least a second lumen, the central probe having a proximal end and a closed distal end, the distal tip having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined distance between the distal and proximal ends of the central probe, the exit port fluidly connecting the second lumen to the exterior of the central probe, c) at least one access line, positioned in the second lumen and slidable within the central probe, an access line having a proximal end positioned in the central probe and a distal end configured to extend outside of the central probe and retract into the second lumen through the exit port, a tip of the distal end of the access line having a shape configured to pierce through tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which a therapeutic portion is injected into the region by the injection probe, d) a ramp, integrally formed with or coupled to an inner surface of the central probe, defining an inner surface of the second lumen, and the ramp is configured to contact and guide the access line to exit the central probe outside of the central probe, e) an electrical connector, which electrically connects the central probe and the access line to the power source, and f) a handle that at least partially houses the electrical connector and is coupled to the proximal end of the central probe and the proximal ends of the injection probe and the access line to facilitate a penetration depth of the injection probe and the distal end of the central probe. The method further comprises the following steps: b) Inserting a central probe into diseased cells in a region of target cells, c) actuating and extending an access line from a central lumen in an axial direction of the central probe, a tip of a distal end of the access line having a needle shape that pierces through the tissue and forms an opening through which at least a portion of the access line enters the tissue and creates a fluid channel through which a therapeutic portion is delivered, d) actuating a ramp integrally formed with or coupled to an inner surface of the central probe, the ramp contacting the access line and directing a trajectory of the access line through an exit port toward a distal end of the central probe, the exit port fluidly connecting the central lumen with an exterior of the central probe, e) piercing the tissue with the access line and creating an opening through which at least a portion of the access line enters the tissue to create a fluid channel for delivery of the therapeutic portion to the tissue, f) retracting the access line into the central lumen, g) injecting the therapeutic portion into the central lumen and delivering the therapeutic portion to the tissue through the fluid channel.

The method further comprises: a) The device is rotated for delivery at least once and the tissue is pierced with the access line to create an additional fluid channel for delivery of the treatment portion to the tissue, then the treatment portion is injected through the fluid channel and delivered to the tissue.

The method further comprises: a) An electroporation system is provided that includes at least two oppositely charged electroporation electrodes positioned configured to surround a region of a target cell, wherein the electroporation electrodes are adapted to extend from a proximal end to a distal end. The distal tip has a needle shape configured to pierce tissue. The electroporation electrode is adapted to be coupled to a source of electrical power. The method further includes b) contacting the region of the target cell with an electroporation electrode, c) delivering an electrical pulse from the electrical power source to the electrode, and d) applying a pulsed electric field sufficient for electroporation to the region of the target cell from the electroporation electrode.

According to some embodiments, a system for electroporation of cells in an electroporation position of an electric field region in tissue in a subject includes a) an electroporation wand housing. The housing includes i) a first pair of electroporation electrodes, and ii) at least a second pair of electroporation electrodes housed in the wand housing, the first and second pairs of electroporation electrodes configured to be oppositely charged, offset from each other by a predetermined angle, and configured to define an outer perimeter of an electric field region. The system further comprises: b) An electrical power supply configured to supply electrical signals at a plurality of waveforms to the first and second pairs of electroporation electrodes, and c) an electrical connector electrically connecting each of the first and second pairs of electroporation electrodes to the electrical power supply.

In some embodiments, the system further comprises a rod delivery system configured to deliver the treatment portion to the electroporation site, the delivery system comprising at least one injection probe defining a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongate cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the treatment portion to the electroporation site.

In some embodiments, the system comprises two pairs of electroporation electrodes and the angle is about 90 degrees.

In some embodiments, the first pair of electroporation electrodes is configured to receive a first electrical signal represented by a first waveform from the power supply, and the second pair of electroporation electrodes is configured to receive a second electrical signal represented by a second waveform from the power supply.

In some embodiments, the first and second pairs of electroporation electrodes are needles configured to penetrate the skin and contact cells in the electric field region.

In some embodiments, the first and second pairs of electroporation electrodes are non-penetrating contacts.

According to some embodiments, a method for electroporating cells in an electroporation location of an electric field region in tissue in a subject comprises: a) An electroporation system is provided that includes i) an electroporation wand housing comprising 1) a first pair of electroporation electrodes, and 2) at least a second pair of electroporation electrodes housed in the wand housing, the first and second pairs of electroporation electrodes being oppositely charged, offset from each other by a predetermined angle, and configured to define an outer periphery of an electric field region, ii) a power supply configured to supply electrical signals at a plurality of waveforms to the first and at least second pairs of electroporation electrodes, and iii) an electrical connector electrically connecting the pair of electroporation electrodes to the power supply. The method further comprises: b) Contacting the electroporation wand housing to the tissue such that an electric field region is between the pair of electroporation electrodes, c) applying a first signal at a first waveform from the power supply to the first pair of electroporation electrodes and a second signal at a second waveform from the power supply to the second pair of electroporation electrodes, wherein the first waveform has a predetermined phase difference from the second waveform, d) applying a pulsed electric field from the first pair of electroporation electrodes to the electric field region, the pulsed electric field being based on the first signal, wherein the pulsed electric field and each subsequent pulsed electric field of the first pair of electroporation electrodes have a voltage and duration below a minimum threshold for electroporation, e) applying another pulsed electric field from the second pair of electroporation electrodes to the electric field region, the another pulsed electric field being based on the second signal, wherein the another pulsed electric field and each subsequent pulsed electric field of the second pair of electroporation electrodes have a voltage and duration below the minimum threshold for electroporation. The paths of the pulsed electric fields of the first and second pairs of electroporation electrodes intersect at the electroporation site, and the application of each pulsed electric field of the first pair of electroporation electrodes to the electroporation site alternates with the application of each pulsed electric field of the second pair of electroporation electrodes to the electroporation site to add up to a continuous pulsed electric field of sufficient voltage and duration to be applied to cells in the electroporation site for electroporation. Each application of the pulsed electric field of the first pair of electroporation electrodes to tissue adjacent to and outside of the electroporation site alternates with a resting period such that tissue adjacent to and outside of the electroporation site receives a pulsed electric field of the first pair of electroporation electrodes that is alternately turned on and off having a voltage and duration below a minimum threshold for electroporation. Each application of the pulsed electric field of the second pair of electroporation electrodes to tissue adjacent to and outside of the electroporation site alternates with a rest period such that tissue adjacent to and outside of the electroporation site receives an alternating on and off pulsed electric field of the second pair of electroporation electrodes having a voltage and duration below a minimum threshold for electroporation.

In some embodiments, the method further comprises delivering the treatment portion to the electroporation site by a rod delivery system comprising at least one injection probe defining a first lumen, the injection probe extending from a proximal end to a distal end thereof and having an elongated cylindrical shape. The distal end of the injection probe has a needle shape and is open for delivery of the treatment portion to the electroporation site.

In some embodiments, the method further comprises providing a sensor system that senses the capacitance of the cell membrane. The capacitive sensing includes: a) contacting the tissue with at least one pair of capacitive sensing electrodes powered by a low voltage power supply, b) transmitting a low power interrogation signal through the low voltage power supply to the at least one pair of capacitive sensing electrodes to generate a low intensity electric field excitation in the electroporation site, c) sensing a voltage or voltage drop across the cell membrane by a voltage sensor, d) sensing a current across the cell membrane by a current sensor; and e) determining, by the electronic signal processing means, the capacitance of the cell membrane based on the voltage drop and the current across the cell membrane.

In some embodiments, the capacitance of the cell membrane is determined before and between application of the pulsed electric field.

In some embodiments, the method further comprises adjusting a pulse width of the pulsed electric field based on a time constant associated with the thin film capacitance after the determination of the capacitance of the cell membrane between the pulsed electric fields.

In some embodiments, the first and second waveforms have the same wavelength.

In some embodiments, the voltage of the power supply is variable from about 50V to 1000V.

In some embodiments, each pulsed electric field of the first and second electrode pairs has a pulse width that is variable from 1 μ s to 1 ms.

In some embodiments, each pair of electroporation electrodes emits each pulsed electric field in a time period of 1/(number of electrode pairs) of the period of the wavelength of each corresponding waveform.

In some embodiments, the cell is selected from the group consisting of: pancreatic, laryngeal, pharyngeal, lip, throat, lung, kidney, muscle, breast, colon, uterus, prostate, thymus, testis, skin, and ovarian cells.

In some embodiments, the cell is a prostate tumor cell.

In some embodiments, the cell is a mammalian cell.

In some embodiments, the cell is a human cell.

In some embodiments, the pulsed electric field of the first and second pairs of electroporation electrodes ranges from about 200 to 500mV.

In some embodiments, the pulsed electric field of the first and second pairs of electroporation electrodes is applied as from about 1 to about 5 electrical pulses.

In some embodiments, the first and second pulsed electric fields are selected from the group consisting of: square wave pulses, exponential wave pulses, finite duration unipolar vibration wave forms, and finite duration bipolar vibration wave forms.

In some embodiments, the first and second pulsed electric fields comprise square wave pulses.

In some embodiments, the therapeutic moiety is selected from the group consisting of: nucleic acids, polypeptides, and chemotherapeutic agents.

In some embodiments, the chemotherapeutic agent is selected from the group consisting of: bleomycin, cisplatin and mitomycin C.

In some embodiments, the electroporation cuvette housing is comprised of a non-conductive instrument.

In some embodiments, the electrically non-conductive appliance is made of plastic.

In some embodiments, each pair of electroporation electrodes determines a field vector and a current path of the corresponding electric field.

In some embodiments, the first and second waveforms have a predetermined phase difference.

According to some embodiments, a system for Electroporation (EP) of cells in tissue of a subject comprises: a) A trocar and b) an EP device. The trocar includes: i) A cannula extending from a proximal end to an open distal end and defining a first lumen configured to receive an obturator, and ii) an obturator extending from a proximal end to a distal end, the proximal end including a handle mounted thereon, the distal end including a blade configured to pierce through skin, penetrate into a body cavity, and form a pathway through which the cannula may be at least partially inserted into the cavity. An obturator is configured to slide within the first lumen, a distal end of the obturator configured to extend outside of the first lumen through the open distal end of the cannula. The EP device is capable of being slidably mounted and retracted within a cannula to access cancer cells, and includes: i) An anchor extending from a proximal end to a distal end, ii) at least two oppositely charged electrodes retractably disposed at the distal end of the anchor and configured to be positioned surrounding a region of a target cell. The electrode is adapted to be coupled to a generator, receive at least one electrical waveform from the generator, and supply at least one of an excitation signal and an EP pulse. The Ep apparatus further comprises: iii) a central probe retractably disposed at the distal end of the anchor and having an inner surface defining at least a central lumen and extending from the distal end of the anchor, at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the treatment portion to the tissue. The distal end of the central probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to pierce tissue.

In some embodiments, the blade of the obturator is configured to extend outside of the cannula through an opening at the distal end of the cannula.

In some embodiments, the EP device electrode is adapted to extend from a proximal end to a distal end, the distal end having a tip with a needle shape configured to pierce tissue. The electrode is adapted to be coupled to a power supply, receive an electrical waveform from the power supply, and supply at least one of an excitation signal and an EP pulse to a region of a target cell.

In some embodiments, an adaptive control method for controlling an Electroporation (EP) pulse parameter during EP of a cell or tissue using an EP system comprises: a) providing any of the systems for providing adaptive control to optimize EP pulse parameters during EP of cells and tissue using any of the Electroporation (EP) apparatuses described herein, b) initializing EP pulse parameters for performing EP in cells or tissue by an initialization module, the initialized EP pulse parameters being based at least in part on at least one trained model, c) applying voltage and current excitation signals to the cells and tissue by a generator and measuring voltage and current across the cells and tissue corresponding to the applied excitation signals by a measurement apparatus, d) obtaining data from the current and voltage measurements by a controller and processing the data to separate desirable data from undesirable data, e) extracting relevant features from the desirable data by the controller, f) applying at least a portion of the relevant features of the desirable data to at least one trained diagnostic model by the controller, g) estimating EP pulse parameters by the controller based on the results of the applied relevant features of the trained model, wherein the initialized EP pulse parameters are based on the at least one trained model and the relevant features, thereby generating a first EP pulse parameter based on the applied relevant features of the trained model, h) optimizing the EP pulse parameters by the controller.

In some embodiments, the method further comprises predicting, by the controller, subsequent EP pulse parameters after the first EP pulse has been applied using a trained model based on previous EP pulses and a change in at least one of the correlation characteristics between the applied EP pulses.

In some embodiments, the method further comprises generating, by the controller, a diagnostic response based at least in part on the applying, wherein the diagnostic response comprises a) tissue detection, b) tumor type detection, c) needle placement detection, d) co-location detection, and d) cell penetration detection.

In some embodiments, the method further comprises: a) Applying, by a generator, a subsequent EP pulse based on subsequent EP pulse parameters, and b) repeating the applying voltage and current excitation signals, repeating the measuring cell or tissue, repeating the obtaining data and separating desirable data from undesirable data, repeating the extracting relevant features; and repeating the applying until i) a predetermined limit of the number of EP pulse sequences or cycles of EP pulses is reached, or ii) a diagnostic decision of the adaptive control method is terminated in response to a prompt.

Drawings

Fig. 1 is a simple schematic diagram depicting some components of an EP apparatus to apply an electrical pulse for EIS in accordance with the present invention.

Fig. 2 depicts an EP electrode array of 4 electrodes (two pairs or sets) and an EM electrode array of 4 electrodes (two pairs or sets), each attached to a suitable power source via a connector and circuitry. Again, each array is a 4-electrode array, but more electrodes may be used. In addition, in these embodiments using two different electrode arrays, the number of electrodes need not be equal in each case; EP arrays of 4 electrodes and EM arrays of 6 electrodes, etc. may be used.

Fig. 3 depicts a schematic for EIS determination using 4 EP and 4 EME electrodes. Fig. 3 depicts a top view of the electrodes of the device inserted into hypothetical tissue containing blood vessels and irregularly shaped tumors (note that EM can also be applied in non-penetrating electrode devices). For non-limiting exemplary purposes, two sets of electroporation electrodes (EPEs) and two sets of electrochemical impedance spectroscopy electrodes (EMEs) are shown, but other numbers and geometries are contemplated. These electrode sets are shown substantially equidistant in fig. 3, but any number of sets can be used consistent with the invention, as will be appreciated by those skilled in the art.

Fig. 4A and 4B show two embodiments (only a single pair of electrodes is shown for simplicity) for producing different electric field regions using intervening EP electrodes with insulating material. FIG. 4A shows a single pair of electrodes with alternating regions of insulating material and bare electrode; in other words, the electrodes have conductors that are alternately evenly spaced along the length of the electrode, with each conductor separated by an insulating material. Fig. 4B shows a similar set, but in this case each conductor is unevenly spaced, enabling an asymmetric electric field to be generated.

Fig. 5 is a diagram of the hardware architecture of an EP generator for generating pulsed electric fields for EPE pairs a and B. The EP device may be based on a Digital Signal Processor (DSP), microprocessor, field Programmable Gate Array (FPGA), application specific integrated circuit, central Processing Unit (CPU), or any multi-purpose programmable device that accepts analog/digital data as input, processes the input according to instructions stored in memory, and provides an output as a result. The switching sequence routine for electrode pairs a and B is programmed and stored in memory. The data bus may be used to display and modify the pulse parameters. High voltage isolation would allow hardware to be used with a high voltage power supply when plugged into a PC. The low voltage power supply may be used to power all auxiliary circuits, such as EMEs for capacitance and impedance measurements, analog-to-digital converters, digital-to-analog converters, repeaters, DSPs, optical switches, and the like.

Fig. 6A, 6B, and 6C depict three different configurations of EPEs and EMEs. In fig. 6C, a single set of electrodes is used, which are connected to respective EPE and EME power sources via a switching mechanism. The switching mechanism is switched on and off when a small actuation voltage is applied across its control terminals. These switches use coupling mechanisms including electromagnetic, electromechanical, piezoelectric, and opto-electronic mechanisms. In fig. 6A, two sets of electrodes EPE and EME are used, each configured and connected to an appropriate power source. Fig. 6B is similar, except that the EPE and EME are offset from each other by a predetermined angle, depending on the number of each electrode type to be used. In this embodiment, the tissue in the zone can be interrogated in different ways. For example, tissue just against the EPE may experience damage (e.g., tissue in a "kill zone"). In fig. 6B, measuring the impedance (including capacitance) between EME #1 and EME #2 can help determine tissue damage at EPE #1, for example, or alternatively can be used for some sort of "electron tumor tomography," as described more fully below with respect to EM.

Fig. 7A, 7B, 7C, and 7D depict different EPE configurations, but only a single pair of electrodes for simplicity. Fig. 7A depicts a set of non-penetrating solid EPEs applied topically to the surface of the skin. Additional sets of EPEs are not shown, but are included. FIG. 7B depicts a set of solid EPEs penetrating into tissue; in this embodiment, the tip of the EPE is substantially pointed to facilitate insertion into tissue, such as a solid needle tip. In this embodiment, the electric field region is "deeper" in the tissue, e.g., below the surface. This results in a three-dimensional electric field along the length and radial dimension between the electrodes. In general, these penetrating EPEs can range from about 1 to about 20mm, depending on the geometry and physiology of the tissue to be treated. In fig. 7C, the penetrating solid EPE is coated with an insulating (non-conductive) material such that only the distal portion of the electrode is exposed. In the embodiment of fig. 7A, 7B and 7C, the TM delivery system will generally be a needle (not shown) that is inserted less deeply into the EP location between the EPEs. In fig. 7D, the penetrating EPE is hollow, with a lumen for TM delivery and a sharp open tip connected to the lumen. On the left, the penetrating electrode has a portion along the axis that is coated with an insulating material. As will be appreciated by those skilled in the art, when the capacitance measurement is complete, the EPE may be used as an Electrical Measurement Electrode (EME) or there may be a separate set of EMEs, as generally depicted in fig. 6.

Figures 8A, 8B, 8C and 8D depict components of an EP apparatus of the present invention (which all rely on cylindrical needles, but other geometries may be used; and only a single pair of EP electrodes is depicted). Fig. 8A and 8B depict a set of EPEs (second set not shown) with a TM delivery (TMD) system. Fig. 8A shows an EPE and TM delivery system inserted into tissue, where the TMD hollow needs to have an open end, a lumen is used to deliver the TM, and the TM is being delivered in a profiled manner. Fig. 8B shows the underside of the device, which may be in the distal end of the rod. Alternatively, as shown in fig. 8C, the TMD system may include a standard syringe that is manually inserted by the administering physician during the procedure. In this embodiment, the syringe may have an optional needle stop to physically prevent deeper penetration at a depth related to the depth of the electric field region. Fig. 8D depicts a TM delivery needle having multiple openings to deliver a TM. This may be useful when delivering larger biomolecules such as plasmids and antibodies, as larger molecules (which are otherwise generally charged) generally diffuse more slowly in tissues than other molecules. Having multiple delivery gene sites within the EP position can therefore be used to allow a higher percentage of cells in a region to take up the TM. Fig. 8D depicts three openings or ports, but any number may be used. Additionally, fig. 8D depicts an opening on a "side" of the needle, but the opening may be located on any portion of the outer surface of the needle, forming a spiral or other shape.

Fig. 9 is a schematic of an EP apparatus according to the present invention comprising a wand housing having a first pair and a second pair of electrodes.

Fig. 10 is a schematic illustration of a first and second pair of EPEs defining an electric field region and electroporation positions in accordance with the present invention.

Fig. 11 is an illustration of a pair of non-penetrating EPEs according to the present invention.

Fig. 12 is a schematic illustration of the offset angles produced by a plurality of EPE pairs according to the present invention.

FIG. 13 is a graphical representation of a first waveform corresponding to a first pair of EPEs and a second waveform corresponding to a second pair of EPEs, in accordance with the present invention.

Fig. 14A and 14B are illustrations of a continuous pulsed electric field in an electroporation site and an alternating on and off pulsed electric field in the electric field region but outside the electroporation site, according to the present invention.

Fig. 15 is a simple schematic diagram illustrating an adaptive control system for optimizing Electroporation (EP) pulse parameters during Electroporation (EP) of cells in tissue of a subject according to the present invention.

Fig. 16 illustrates an EP system for use in an adaptive control system during Electroporation (EIS) of cells in tissue of a subject according to the present invention.

Fig. 17A is a schematic illustration of an exemplary Electroporation (EP) apparatus with electrodes integrated around a central injection element and a central portion delivery probe for use in an adaptive control system for optimizing EP pulse parameters, and fig. 17B is a bottom view of the EP apparatus.

Fig. 18A and 18B are schematic illustrations of perspective and bottom views of an exemplary Electroporation (EP) device with electrodes integrated around an infusion element and a portion of a delivery probe for use in an adaptive control system for optimizing EP pulse parameters.

Fig. 19 is a schematic of a plurality of electrodes positioned on a helical portion delivery probe of an EP apparatus according to the present invention.

Fig. 20A is an illustration of multiple central injection probes of an EP apparatus according to the present invention, each containing a helical blade for generating a channel, fig. 20B is a schematic illustration of an EP apparatus with the central injection probe of fig. 20A surrounded by multiple electrodes, and fig. 20C is an illustration of a bottom view of fig. 20B.

Fig. 21 illustrates an EP apparatus with a helical center probe and a helical electrode according to the present invention.

Fig. 22A, 22B and 22C are schematic views of EP apparatus with a distal electrode and a central probe according to the present invention.

Fig. 23A, 23B and 23C, 23D, 23E and 23F illustrate various EP devices according to the present invention.

Fig. 24A, 24B and 24C illustrate a trocar-based direct stick applicator EP system according to the present invention.

Fig. 25 and 26 illustrate a catheter/endoscope based EP device according to the present invention.

Fig. 27 is a schematic illustration of an EP apparatus for delivering a therapeutic moiety to a region of target cells of a tissue according to the present invention.

Fig. 28 is a schematic view of an exit port and an access line of a blade tip with the EP apparatus of fig. 27 according to the present invention.

Fig. 29 is a schematic illustration of a ramp and lane-taking line guided by the ramp of the EP apparatus of fig. 27 in accordance with the present invention.

FIG. 30 is a schematic view of an injection probe and a center probe coupled to each other in accordance with the present invention.

Fig. 31 is an illustration of a curved lane line of the EP apparatus of fig. 27 according to the present invention.

FIG. 32 is a schematic illustration of a Capacitive Sensing (CS)/EIS sensing system according to the present invention.

Fig. 33 is an illustration of a method for delivering a therapeutic moiety to a region of target cells of a tissue using an EP device according to the present invention.

Fig. 34A, 34B and 34C illustrate various configurations of EP apparatus (of fig. 27) according to the present invention. In fig. 34A, multiple lane lines are used, all deployed at once, resulting in a "star" pattern. In fig. 34B, a single lane line is used that is deployed, retracted, rotated, and re-deployed, producing the same "star" pattern but sequentially. In fig. 34C, a single channel wire is used, but after deployment and retraction of the channel wire, the rod housing is slightly removed and the channel wire is again deployed, forming a "comb" structure.

Fig. 35 shows a housing containing a curved channel wire that remains rigid when in place, but after deployment of the wire, returns to its curved shape and forms the curved channel of the fluid reservoir.

Fig. 36 is a flow diagram illustrating a control routine of an adaptive control method for controlling EP pulse parameters during use of an EP system in accordance with the present invention.

Fig. 37 is a flow diagram illustrating a further-ahead feedforward control routine for optimizing EP pulse parameters using the control routine of fig. 36 in accordance with the present invention.

FIG. 38 is a graphical representation of an initial training phase of a model to estimate pulse parameters in accordance with the present invention.

FIG. 39 is a graphical representation of a trained model used to estimate first pulse parameters (initialization) in accordance with the present invention.

Fig. 40 is a flow diagram illustrating an EP diagnostics routine in a method for adaptive control of EP pulse parameters in accordance with the present invention.

Fig. 41A and 41B are flowcharts illustrating a method for adaptive control of EP pulse parameters according to the present invention.

Fig. 42A illustrates the distribution of the percentage applied electric field across the lipid bilayer versus the time constant, fig. 42B illustrates the distribution of the time constant measured before EP, fig. 42C illustrates the effect of modulating the pulse width based on pre-pulse EIS data, where the pulse duration is set at a multiple of the time constant for each tumor, and fig. 42D illustrates data showing the relative change in the calculated time constant after EP with respect to the resulting luminescence according to the present invention.

FIG. 43 illustrates model fitting parameters for normal C57BL/6J mice and transgenic PDGF-C mice, where the parameters expressed are the calculated time constants of (A) solution resistance, (B) admittance, (C) Constant Phase Element (CPE), and (D).

Figure 44 illustrates a histogram of the percent decrease in solution resistance after injection of plastid DNA.

FIG. 45 illustrates luminescence data observed 48 hours after intratumoral EP with 50. Mu.g of plastid DNA expressing luciferase. The EP conditions were set at 500V/cm, 8 pulses were applied, and the duration was set at a multiple of the calculated average time constant.

FIG. 46: luminescence data observed 48 hours after intratumoral EP at 50 μ g of plastid DNA expressing luciferase. EP conditions were set at 350V/cm, 8 pulses were applied, and the duration was set at a multiple of the calculated time constant for each individual tumor.

FIG. 47: luminescence data plotted as a function of the calculated time constant after electroporation. Longer pulses resulted in a decrease in the calculated time constant, with greater than 20% of the groups being significantly different from the control. The short pulses cause an increase in the calculated time constant.

FIG. 48: an equivalent circuit model of the CPE-based organizational model.

Detailed Description

I. Overview

The present invention is generally directed to devices, systems, and methods having improved and desirable optimization of Electroporation (EP) pulse parameters for use in EP of cells and tissues of a patient. As further described herein, the present invention has a variety of uses, including but not limited to, for example, the ability to insert therapeutic moieties (including small molecule drugs, plasmids encoding therapeutic proteins, etc.) into cells. The invention is particularly suitable for oncology applications. The present invention allows for the determination of appropriate EP conditions and/or EP protocols in real time using Electrical Measurements (EM), including but not limited to Electrochemical Impedance Spectroscopy (EIS). The present invention aims to improve the EP process by integrating a feedback control mechanism. Thus, the systems and methods of the present invention may be used with any EP apparatus/applicator and any method, such as those outlined in U.S. provisional patent application nos. 62/214,807, 62/214,872, 62/141,142, 62/141,182, 62/141,256, and 62/141,164, all of which are expressly incorporated herein by reference in their entirety, specifically including the drawings, illustrations, and descriptions of the drawings and components therein.

The EP parameters currently used in clinical trials were established empirically in preclinical mouse studies using homogeneous syngeneic tumor models. Typically, the electrical parameters are selected to give the highest rise via average expression of the injected electroporated nucleic acid alone. Previous studies in the field have analyzed the effect of pDNA concentration, electric field (e-field) strength, pulse, tissue type, electrical conditions, injection volume, molecules of interest, concentration, and applicator geometry on expression. Each of the foregoing parameters has been determined to significantly affect the resulting expression.

In order to maximize the efficacy of EP, a quantifiable measure of film integrity, which can be measured in real time, is desirable. Electrochemical Impedance Spectroscopy (EIS) is a method for the characterization of physiological and chemical systems and can be performed with standard EP electrodes. This technique measures the electrical response of the system over a range of frequencies to reveal energy storage and dissipation properties. In biological systems, the extracellular and intracellular substrates resist electrical currents and can therefore be represented electrically as resistors. The lipids of intact cell membranes and organelles store energy and are represented as capacitors. The electrical impedance is the sum of these resistive and capacitive elements over a range of frequencies. To quantify each of these parameters, the tissue impedance data can be fitted to an equivalent circuit model. Real-time monitoring of the electrical properties of the tissue will enable feedback control of EP parameters and lead to optimal transfection in heterogeneous tumors. Using EIS feedback will allow (1) real-time adjustment of delivery parameters, (2) delivery of only the pulses necessary to produce a therapeutic response, and (3) thus reducing overall EP-mediated tissue damage.

Various embodiments of the present invention are directed to providing a closed loop EP control system that optimizes the EP process with tumor-specific measurements taken before and between each EP pulse using feedback based on tissue sensing. Tissue sensing is used to measure the membrane charge time for a particular tumor to adjust each EP pulse for optimal treatment.

As one of ordinary skill in the art will appreciate, successful EP occurs when the cell membrane breaks, resulting in a change in capacitance. Thus, by monitoring and measuring electrical properties, such as impedance (including capacitance) before, during and/or after EP pulses, relevant empirical data can be collected during an initial training phase and used to generate a model.

Various embodiments of the present invention are directed to adaptive control methods and systems for improving or optimizing controlled EP pulse parameters during EP of cells and tissues using the aforementioned closed-loop EP control systems and devices.

In some embodiments, the control system may include a measurement device, an initialization module, a signal generator, a controller, and a memory module. The control method described herein is implemented in the control system.

In one aspect, the measurement device measures tissue/cellular conditions, such as dielectric and conductive properties of cells and tissues. The measurement device may comprise one or more different measurement devices to facilitate measurement of tissue/cell conditions. For example, the measurement device may include a voltage sensor/device and/or a current sensor/device. The voltage sensor may be configured to measure a voltage across the cell or tissue when the excitation signal and/or EP pulse is applied to the cell or tissue. The current sensor measures the current across the cell or tissue when the excitation signal and/or EP pulse is applied to the cell or tissue. The results of the measurements (e.g., measured data) may be sent to the controller for further processing.

The initialization module may be configured to initialize EP pulse parameters for performing EP on cells and tissue. The EP pulse parameters may be predetermined EP pulse parameters that are empirically established based on prior experimental/clinical trials. Alternatively, the predetermination of EP pulse parameters may be based at least in part on one or more trained models. The signal generator may generate an excitation signal and/or electroporation pulses that are applied to the cells and tissues. The measurement device measures tissue/cellular conditions, such as dielectric and conductive properties of cells and tissue, in response to application of the excitation signal and/or electroporation pulse.

As mentioned, the controller receives measured data, which corresponds to the results of the measurement of the tissue/cell condition. The controller then processes the measured data to facilitate diagnosis/identification of characteristics of the tissue and cells, and/or to determine updated control parameters for the system. For example, the heterogeneity or homogeneity of the tissue can be assessed. The controller may include (in any combination) a pre-processing module, a feature extraction module, a diagnostic module, and a pulse parameter estimation module.

The pre-processing module obtains measured data from the measurement device and pre-processes the measured data to separate desirable data from undesirable data. For example, undesirable data may include noise, direct current bias. Preprocessing may include scaling the measured data based on a normalized value, such as a standard deviation, performing digital filtering of the measured data, and validating the measured data.

The feature extraction module extracts information, such as relevant features, from the desired data. The relevant features may be quantitative information. For example, quantitative information may be extracted using the calculation routines described herein. The relevant characteristics of the desired data are sent to a diagnostic module for further processing. For example, the diagnostic module applies at least a portion of the desired relevant features to one or more trained diagnostic models to determine whether the next step is to select the next applied EP pulse parameters or to stop the control process if a diagnostic problem is detected, such as the electrode not being placed in tissue. The pulse parameter estimation module is configured to select or generate a next applied EP pulse parameter based on the results of the diagnostic module and the feature extraction module.

In some embodiments, the present invention relates to "one step ahead feedforward control". By "step-ahead feed-forward control" is meant that prior to application of the first EP pulse, the parameter estimation routine initializes initial control parameters for the first pulse based on a model trained in an initial training phase using empirical data from previously performed experiments. These previously performed experiments may be based on, for example, tissue samples with tumors having similar characteristics as those of the current tissue to be subjected to the control method of the present invention. For example, the type, size, or location of a melanoma tumor may be used to build a data set to serve as the basis for an initial model. The initial excitation signal, which includes voltage and current signals, is applied by a signal generator (e.g., a proprietary signal generator as described herein). The measurement device measures the response of the tissue to the excitation signal. The controller derives "features" based on the measurements and uses a trained model to compare the extracted features to old features derived from empirical data obtained in previous experiments. The old and derived "features" are obtained from tissue sensing measurements such as EIS. The model may be trained based on the tissue or tumor type identified by the diagnostic module in the diagnostic phase and then used to select the optimal parameters/conditions for the first EP pulse. These first pulse parameters are thus "fed forward" to be applied as first pulses for the control routine, in contrast to conventional EP systems and methods in which the parameters/conditions of the first pulses are based on fixed or static conditions. In this sense, the method of the present invention utilizes feed forward control to provide optimal EP parameters based on the sensed tissue type, in conjunction with feedback control to sense cellular conditions, such as the degree of penetration, and adjust the pulse parameters accordingly.

Changes in tumor characteristics such as tumor location, size, and the degree of angiogenesis, fibrosis, and necrosis that generally affect the outcome of treatment, lead to poor predictability of effective EP conditions for gene delivery, and thus variable outcome of treatment. Conventional EP systems apply open-loop control systems using static parameters that rely on a priori knowledge as determined by preclinical studies in homogeneous syngeneic tumor models. However, preliminary data have shown that even in homogeneous tumors, the time required to apply an electrostatic field across the cell membrane follows a log-normal distribution. Even in homogeneous models, applying static parameters to different tumors results in a wide range of applied electrostatic fields across the cell membrane and in treatment variability. The present invention overcomes the aforementioned deficiencies of the prior art by implementing a control method that employs a closed-loop control system that uses feedback based on tissue sensing to optimize the EP process with tumor-specific measurements taken before and between each EP pulse. Thus, by using EIS feedback control in combination with "one step ahead feed forward control", the present invention is able to more effectively predict effective parameters for EP taking into account changes in tumor characteristics that typically affect treatment.

Electrochemical Impedance Spectroscopy (EIS)

The systems and methods of the present invention may comprise Electrochemical Impedance Spectroscopy (EIS) (or tissue sensing) measurements, which may be performed using EP devices. In some embodiments, the EP device may include an electroporation electrode (EPE) for applying EP pulses and an Electrical Measurement Electrode (EME) for applying a low voltage interrogation signal to the cells. In some embodiments, the electrodes of the EP device serve as both EMEs and EPEs, and a solid state relay may be used to switch between a high voltage EP pulse circuit and a low voltage EIS interrogation circuit, as illustrated in fig. 1. FIG. 1 is a simple schematic depicting some components of an EP device used to apply electrical pulses for EIS according to the present invention. Although an electrode array of 4 electrodes is shown, this is not limiting, where the array of electrode pairs contains 2, 4, 6, 8, 10 and 12 or more electrodes, all suitable for use in the present invention. Further, while the electrodes are shown as having a straight shape, this is not limiting as the electrodes may have a curved or spiral shape, as will be described below with respect to various EP devices that may be used in the systems and methods of the present invention. Fig. 1 depicts the situation where the electrodes of the array act as EP electrodes when connected to an EP circuit or as Electrical Measurement (EM) electrodes when connected to a tissue sensing/EIS circuit. As discussed herein, when the electrodes of the EP device serve as both EME and EPE, the electrodes are switched between EPE and EME modes by the relay switch. That is, the solid state repeater is used to switch between a high voltage EP pulse circuit and a low voltage EIS interrogation circuit, as illustrated in fig. 1. That is, the proprietary generator of the present invention is able to supply both high voltage pulses and low voltage interrogation signals to the EP device when necessary. In other embodiments where the EP device is provided with separate EPEs and EMEs, the EP device may be connected to both power sources via a switching mechanism that is switched on and off when a small actuation voltage is applied across its control terminals. These switches use coupling mechanisms including electromagnetic, electromechanical, piezoelectric, and opto-electronic mechanisms.

Due to the general knowledge about the electroporation conditions, the capacitance and resistance measurements taken prior to the application of the EP pulse enable a priori knowledge of the conditions that will cause instability of the capacitive elements, e.g., cell membranes. Measuring capacitance between pulses allows the electrical condition to be adjusted based on time constants associated with the thin film capacitance and resistance, including pulse width (which can be calculated from the associated time constants). In addition, this information allows the process to be stopped when a desired decrease in time constant is reached, for example when the membrane integrity has been compromised, thus allowing the introduction of a therapeutic moiety.

In some embodiments, the EP device uses a different set of EPEs and EMEs, as generally depicted in fig. 3. In some cases, as more fully outlined below, when different sets of EPEs and EMEs are used, the EMEs and EMEs can shift, allowing for impedance measurements in different regions of the electric field region, as generally discussed below and in the legend of fig. 3. In these embodiments, an additional low voltage EME power supply is used in addition to the higher voltage EPE power supply, along with appropriate circuitry and connectors.

In other preferred embodiments, as described above, and as will be used to illustrate the system and method of the present invention, the EP device uses a single set of electrodes for both EP and EIS measurements. EIS measurements can be performed using EPEs without adversely affecting tissue properties. It is desirable to perform low power EIS measurements and high power EP pulses using the same electrode, as this reduces the number of electrodes required and directly measures tissue response. EIS is a low power technology that enables real-time monitoring of an organization. This technique is performed by applying a series of low voltage excitation signals across a pair of electrodes and measuring the response current over a range of frequencies. The magnitude and phase of each applied excitation is then calculated and fitted to an equivalent circuit model of the tissue as explained below (as shown in fig. 48), hereinafter referred to as the "CPE-based tissue model".

The impedance measurements may be obtained using the following equation:

in the above equation, Z (f) is the tissue impedance in ohms and f is the frequency in Hertz; j is a number

A constant of (d); q ₀ Admittance in siemens (at f =1 Hz); r _s Resistance in ohms; and α is a unitless Constant Phase Element (CPE).

As illustrated in the model, the resistive element (R) _I And R _E ) Due to the intracellular and extracellular matrix, respectively, and the lipid structure is composed of Constant Phase Elements (CPE) of tissues and cells _M ) And (4) showing. CPE (customer premises Equipment) _M Is a charge or capacitance (represented by Q) representing the lipid bilayer _M Represented) and a function of a scalar (represented by a) ranging from 0 to 1 representing the non-ideal properties of the capacitor. As will be discussed further below, the time constant for charging the lipid bilayer may then be calculated as τ = (R) _I Q _m ) ^1/α . Calculating the time constant in this manner is integral to the method of the present invention in that the time constant is then used to identify the optimal EP pulse duration before, during, and/or after each treatment.

By using an array of EPEs and/or additional electrical measurement electrodes EME, the tissue in the area enclosed by the electrode array can be interrogated. This information can be used to guide, for example, EP conditions. That is, the input signal, e.g., chirp (or many other signals as outlined below), is interrogated using a different EIS, and the output signal allows the device to be fitted to a tissue model to determine the properties of the tissue and EP signal to be used. For example, referring to fig. 3, after insertion of the electrodes, different interrogations can be run. For example, comparing the impedance between

electrodes

1 and 2 and 1 and 8 can help determine that the tissue between

electrodes

1 and 8 is "normal" tissue, rather than "abnormal" or "diseased" tissue between 1 and 2. Similarly, interrogation between

electrodes

7 and 8 or 6 and 7 can help determine that electrode 7 is near or in a blood vessel and therefore should not be used for electroporation. Thus, for example, these measurements can be used to solve the following four problems, as well as any other relevant queries that depend on the data necessary based on the scope of the experiment.

1) Is each electrode in good contact with the tissue? As one of ordinary skill in the art will appreciate, the use of electrodes in difficult to access areas or on certain compliant skins can lead to uncertainty with adequate insertion of both electrodes into the tissue to be treated. This results in a heterogeneous electric field and poor delivery. 2) Is the electrode inserted into viable tissue? The electrodes are inserted into abnormal tissue, and certain tumor tissue may be heterogeneous in texture and/or cellular integrity, etc., with many tumors having necrotic and/or apoptotic cellular regions. The electrodes are inserted into positions that may not result in a good and/or even an electric field and therefore may not be used in the procedure of the invention. 3) Is the Treatment Moiety (TM) or drug in the correct position? In this embodiment, this measurement can be made before and after injection of the TM solution, and the difference can tell whether more TM solution should be injected. 4) There are electrodes that should not be used due to their location and/or contact? Again, referring to fig. 3, these EIS measurements may allow for the determination of electrode 7 placement irregularly (e.g., in or near a blood vessel, etc.), or the location of electrode insertion resulting in poor electrical contact due to tissue heterogeneity and/or integrity. 5) Is the tissue (e.g., tumor) adequately electroporated? This is the same as tissue sensing, as it measures the integrity of the cell membrane. Thus, these measurements can be taken before EP (to establish a baseline), during EP, and after EP to ensure that EP does occur.

In addition, these EIS measurements can be used to determine ideal EP conditions for providing improved or optimized EP pulse parameters as will be described below with respect to the adaptive control method of the present invention. In some embodiments, the methods of the present invention may comprise contacting tissue in electric field region 100 or in electroporation position 110 (shown in fig. 10) with a pair of EPEs/EMEs 120. A low voltage power supply electrically connected to the EPE/EME is used to apply a low voltage interrogation signal to the EPE/EME. Methods for sensing impedance and/or capacitance may include, but are not limited to, waveforms such as phase locked loops, square wave pulses, high frequency pulses, and chirps. The voltage and current sensors are used to sense the voltage drop and current flowing through the circuit, and these parameters can then be processed by the controller as illustrated in fig. 1 to determine the average impedance of all cells in the measurement area.

As described above, capacitance and resistance measurements are indicators of the health of the cell and can be used to determine how long an electrical pulse is applied in order to disrupt the cell membrane and provide sufficient conditions for electroporation. Once the average impedance of the cells has been determined, it is possible to determine several characteristics of the measured cells, including but not limited to the initial condition of the cells or tissue, such as whether the cells are diseased (indicated by a lower than average capacitance), whether the cells are healthy, the positioning of the electrodes-whether the electrodes are properly positioned around the area around the tissue/cells of interest and/or whether the electrodes are in the correct position for effective electroporation, and the time constant, as briefly discussed above for the cells (further described below).

In some embodiments, impedance measurements may be made across several EI sensing electrode pairs to determine whether the average of all cells in the electric field region 100 is consistent and for more accurate readings of a particular location. If the EI measurements are inconsistent across several electrode pairs, this may indicate an inconsistency in the homogeneity of the cell, thus requiring different time constants to be applied to different sets of electrodes. The time constant gives an indication of the pulse width that will be applied to the cell for electroporation to occur. Charging the capacitor to its maximum (i.e., the case where the capacitor/cell cannot store energy due to electroporation, where good transfection occurs) takes about a 5-fold time constant. It is thus possible to determine the pulse width for charging the capacitor to a point just before electroporation occurred, and thus to determine the charging of the capacitor for at least 5 times the time constant (τ) _C ) The necessary pulse width. After determining the time constant, the pulse width is set for each set of EPEs accordingly, based on the time constant determined for the cells in the area encompassed by the EPE.

The time constant can be based onA circuit model as illustrated and described herein and derived from a series of equations below. The time constant for the purposes of the present invention is described as the potential (V) applied across the terminals _a ) Drive CPE to half of the applied potential (V) _CPE ＝V _a /2) the amount of time required (τ).

(1)

(2)

(3)

(4)τ＝(R _s Q ₀ ) ^1/α

Here, | V _CPE I is the voltage across the CPE, and V _a Is the voltage applied across the terminal or membrane. At (2), | V _CPE I quilt | V _a I/2 substitution, thus leading to step (3) of calculating f, and f is substituted

And replaced to derive the final time constant equation used in the calculation of the ideal pulse width for the EP pulse.

Thus, the method and system of the present invention utilizes electrical-based measurements and feedback to substantially improve the EP process, as will be further described below. Since the EPE serves as both an EPE and an EME, feedback is provided by the EPE, so no additional hardware is required. Fitting the electrical data to the modified Randles model circuit allows for parameter monitoring of the condition of the thin film. Tumor tissue can thus be fitted to the modified Randles equation in real time. The modification involves replacing the Constant Phase Element (CPE) with a capacitive/resistive element. CPE provides a realistic representation of the film, where Q = admittance; and alpha is more than or equal to 0 and less than or equal to 1.

Fig. 10 is a schematic illustration of a first and second pair of EPEs defining an electric field region and electroporation positions in accordance with the present invention. Fig. 14A and 14B are illustrations of a continuous pulsed electric field in an electroporation site and an alternating on and off pulsed electric field in the electric field region but outside the electroporation site, according to the present invention. As will be appreciated by those skilled in the art, successful electroporation occurs when the cell membrane breaks, resulting in changes in capacitance and resistance. When subjected to an electric field, the cells generally act as capacitors. When the electric field is applied for a sufficiently long period (depending on the cell properties, health, size, etc.), charge accumulates at the cell membrane until it reaches a certain threshold and causes disruption of the membrane integrity. In embodiments where the EPE and EME are different electrodes, the EME may be powered by a low voltage interrogation circuit. The present invention also includes voltage and current sensors, as illustrated in fig. 15, to measure the current and voltage across the cell membrane and tissue, and a controller processes the voltage and current to determine the average capacitance of the cells in the electric field region 100.

Impedance may be measured based on charge redistribution in the cell in response to low frequency electric field excitation of the low voltage interrogation circuit. Impedance can be measured before, during, and after application of the electroporation electric field to determine cell conditions, including but not limited to cell health, placement of electrodes relative to the cell for optimal electroporation, and most importantly a time constant, which can be used to determine the pulse width of the electric field to be applied to the cells in the electric field region. As described previously, in general, it takes five periods of time constant to charge the capacitor to its maximum value, i.e., just before electroporation occurs, so the pulse width of the initial electroporation electric field pulse can be set to 5 times the time constant. This pulse width is insufficient to cause electroporation in cells outside of electroporation site 110, as described above, but sufficient to cause electroporation in cells of tissue in electroporation site 110 that are subjected to the additive effect of electric fields from all sets of EPEs being applied as one continuous electric field. The impedance measurement may be applied again after the first EP electric field has been applied, and the percentage decrease in impedance or time constant may be calculated and compared to a predetermined value to determine whether the cells in the electroporation site have been sufficiently electroporated. If not, the system and method of the present invention adjusts the pulse width of the pulsed electric field through the next collective electroporation based on the calculated percentage drop in capacitance until it is determined that sufficient EP has occurred in the EP location. Thus, impedance measurements between pulses allow the electrical condition, i.e., pulse width, to be adjusted based on the time constant associated with cell membrane capacitance and resistance, and the electroporation process can be stopped when the desired support of capacitance, time constant, or film integrity is reached.

Various embodiments of the present invention are directed to control systems and methods for electroporating cells in EP locations of tissue using the various electroporation devices of the present invention described herein.

Adaptive control system of the invention

Various embodiments of the present invention are directed to systems implemented in Electroporation (EP) devices for providing adaptive control to optimize controlled parameters during EP of cells and tissues. In some embodiments, as illustrated in fig. 15 and 16, the adaptive control system comprises a measurement device configured to measure dielectric and conductive properties of cells and tissues.

Examples of dielectric and conductive properties may include capacitance, resistance, and impedance. In some embodiments, the measurement device includes a voltage sensor configured to measure a voltage across the cell or tissue resulting from each of the excitation signal and each EP pulse applied to the cell or tissue, and a current sensor configured to measure a current of the cell or tissue resulting from each of the excitation signal and the at least one applied EP pulse. Voltage and current measurements are indicative of and used to calculate various dielectric and conductive properties of cells and tissues.

Fig. 15 is a simple schematic diagram illustrating an adaptive control system for optimizing Electroporation (EP) pulse parameters during Electroporation (EP) of cells in tissue of a subject according to the present invention, and fig. 16 illustrates an EP system for use in the adaptive control system during Electroporation (EIS) of cells in tissue of a subject according to the present invention. In some embodiments, as illustrated in fig. 16, the EP system of the present invention comprises (a) an EIS-equipped EP generator with data recording (e.g., generator 1530 of fig. 15), (B) a graphical user interface for programming pulse conditions, setting feedback criteria, and downloading EIS and pulse performance characteristics, (C) a proprietary applicator (EP device) consisting of two electrodes surrounding a central injection lumen, and (D) a foot pedal switch to remotely activate the EP process. While the EP system of fig. 16 illustrates one type of EP device, it should be understood that the control system of the present invention may be incorporated into any EP device described herein to perform the adaptive control method described herein.

In some embodiments, the adaptive control system includes an initialization module 1520 configured to initialize EP pulse parameters for performing electroporation in cells or tissue. EP pulse parameters may include, but are not limited to, voltage magnitude, repetition rate, and pulse width. The initialized EP pulse parameters are based at least in part on the at least one trained model. The trained model may be, but is not limited to, a physics-based model, an empirical model, or a data-driven model. EP pulse parameters may include, but are not limited to, pulse width, number of pulses, amplitude/field strength, and frequency.

In some embodiments, the adaptive control system further comprises a signal generator 1530 configured to generate the excitation signal and deliver the EP pulse to the cells and tissues by (EPE/EME). The signal generator 1530 may be an EIS-equipped pulse generator that provides an initial excitation signal at a predetermined pulse width based on experimental data observed offline, i.e., data in previous electroporation experiments performed with respect to tissues/cells having similar properties to those to be subjected to the control method of the present invention. In some embodiments, signal generator 1530 is capable of supplying both a low voltage excitation (interrogation) signal as well as a high voltage signal for EP pulses. An example of such a generator is the proprietary generator (a) illustrated in fig. 16. The generator is capable of performing real-time feedback control based on EIS data before and between each EP pulse. The generator can output a minimum of 10V and a maximum of 300V with a pulse duration ranging from 100 mus to 10 ms. The EIS data captured before and between pulses were obtained by 10 data points acquired by the generator at each decade over the range of 100Hz to 10 kHz. The acquisition of EIS data over this spectrum is achieved in 250ms, which is fast enough to: (1) Executing a routine to determine a time constant for a next pulse; (2) storing the EIS data for post-analysis; and (3) does not interrupt clinically used EP conditions. The generator may be capable of a minimum output load impedance of 20 ohms and a maximum load impedance of an open circuit. The custom generator interfaces with a variety of standard EP device applicators and is capable of supporting up to 6 electrodes. A solid state repeater may be used to switch between a high voltage EP pulse circuit and a low voltage EIS interrogation circuit. To allow hands-free operation of the generator, a foot pedal may be added to trigger, pause or abort the EP process.

A measurement device 1510 comprising a voltage sensor and a current sensor measures voltage and current across cells and tissue in response to application of the excitation signal and/or EP pulse. In some embodiments, the measurement device is incorporated into the electrodes of EP device 1540 of the present invention, but is not so limited. In other embodiments, the measurement device may be separate from the electrodes and implemented elsewhere in the control system.

In some embodiments, the control system of the present invention includes a controller 1505 configured to receive sensor data from a measurement device corresponding to the results of measured cell or tissue properties (i.e., dielectric and conductive, such as capacitance, resistance, and impedance) and process the data into diagnostics and updated control parameters. In some embodiments, the controller consists of four modules, including a pre-processing module 1550, a feature extraction module 1570, a diagnostic module 1580, and a pulse parameter estimation module 1560. The pre-processing module obtains data from the current and voltage measurements and pre-processes the data to separate desirable data from undesirable data. Undesirable data may include, but is not limited to, outliers, out-of-range values, and missing values. Data gathered from EIS measurements is fitted to a tissue impedance model, i.e., the CPE-based tissue model described above, in real time using a controller, such as a microprocessor with a reduced instruction set computing architecture.

In some embodiments, the feature extraction module extracts quantitative information from the consensus data using a calculation routine. The calculation routines may include, but are not limited to, linear curve fitting parameters, non-linear curve fitting parameters, cross-correlation, curvature, mean, average, median, range, standard deviation, variance, and kurtosis. When operating in a feedback mode, the measured characteristics of the EIS data may be used to control parameters associated with the EP process.

In some embodiments, the diagnostic module applies at least a portion of the relevant features of the desired data to at least one trained diagnostic model. Together with a diagnostic model of the relevant features, are used to make decisions on the applied pulses. The diagnostic module may combine several features to identify whether there is proper needle placement of the EP device, whether a drug or gene is located between EPE pairs, whether an EP pulse is effectively applied to the transfection, and whether another pulse can be applied to the same electrode pair.

In some embodiments, the pulse parameter estimation module is to generate next-applied EP pulse parameters based on results of the diagnostic module and the feature extraction module. In some embodiments, the control system of the present disclosure further includes a memory module to store processed device/sensor data and the trained model for feature extraction of the controller.

Electroporation devices and methods

EP electrode configuration

The EP apparatus of the present invention is applied to two main treatment areas: delivery of therapeutic moieties and tissue electroporation/ablation. In general, and for many of the embodiments outlined herein, a patient has a disease, such as cancer, localized in a particular tissue that would benefit from intracellular delivery of a Therapeutic Moiety (TM). Alternatively, in some embodiments, it is desirable to kill a minigene site of cells within a tissue (sometimes referred to as "irreversible electroporation" or "electroporation denudation" in the context of electroporation). As is known in the art, one advantage of irreversible electroporation is that it results in apoptosis rather than necrosis as is the case with other common ablation techniques. While most of the discussion herein is relevant to the former, FPA systems and methods in the absence of TM delivery are always contemplated.

The EP device and method of the present invention are used to electroporate cells in the tissue of a patient or subject and deliver TM to the electroporation site for treatment thereof. Generally, the EP apparatus of the present invention is used to treat diseased or abnormal tissue, such as cancerous tissue. The term "cancer" encompasses a large number of diseases characterized generally by inappropriate, abnormal, or excessive cell proliferation. Examples of cancer include, but are not limited to, breast, colon, prostate, pancreas, skin (including melanoma, basal cell and squamous cell), lung, ovary, kidney, brain or sarcoma. Thus, cancerous tissue, including skin tissue, connective tissue, adipose tissue, and the like, can be treated using the system of the present invention. These cancers may be caused by chromosomal abnormalities, degenerative growth and development disorders, agents that promote cell division, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of genes, alterations in gene expression, or carcinogenic agents. The term "treatment" includes, but is not limited to, inhibition or reduction of cancer cell proliferation, destruction of cancer cells, prevention of cancer cell proliferation or prevention of malignant cell initiation, or suppression or reversal of progression of transformed premalignant cells to a malignant disease, or amelioration of the disease. The term "subject" or "patient" refers to any animal, preferably a mammal such as a human. Livestock use is also intended to be encompassed by the present invention.

The systems and methods of the present invention are suitable for electroporation of cells in tissue. The terms "electroporation," "electroosmosis," or "electrokinetic enhancement" ("EP") as used herein interchangeably refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biofilm; its presence allows the delivery of therapeutic moieties (including but not limited to biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water) from one side of the cell membrane to the other. By application of an electric field over a period of time, vm =1.5 × radius × E according to the formula _ext The cell membrane accumulates charge and generates a voltage across the membrane, where the radius is the radius of the cell, and E _ext Is the external electric field of the cell. Generally, cell membranes rupture (e.g., form pores) at approximately one volt, but in addition to the placement of cells in an electric field, there can be differences in the size and shape of the cells. For example, the myocyte cells span the width of the cell and follow itThe length ratio has a higher capacitance. Similarly, larger cells are generally electroporated at lower voltages. As discussed herein, the use of the EM or capacitive sensing techniques of the present invention can help optimize EP pulses and durations by determining bulk properties of cells in an electric field.

"electroporated cells" include those cells having a transiently open pore in the cell membrane that closes upon dissipation of charge on the cell membrane ("open pore cells"), as well as those cells that have undergone electroporation such that the cells now contain an exogenously added therapeutic moiety and have a closed pore (e.g., are again intact).

Referring to fig. 9, an electroporation device according to various embodiments of the invention is illustrated and generally designated by the numeral 10. The electroporation device 10 may generally include an electroporation wand housing 12, optionally in the form of a cylindrical tube (although other geometries may be used), a first pair of electroporation electrodes a and at least a second pair of electroporation electrodes B housed in the wand housing. The wand housing may optionally contain other components, including systems for TM delivery, switching circuitry, and the like.

In some embodiments, the wand housing is shaped for convenient use by a physician, such as with a molded handle portion or grip, optional illumination elements at the distal end, a camera for viewing and recording the treatment site, biopsy forceps, tissue scissors, an engagement device, a suturing system, and the like.

By "electroporation electrode pair", "EPE" herein is meant a pair of two electrodes which are configured to be oppositely charged when connected to a power supply. The first and second pairs of electroporation electrodes may be stationary or retractable within the electroporation wand housing 12. The electroporation wand housing 12 may further include a circuit board 16 having a plurality of sliding sockets through which the electroporation electrodes A and B slidably retract and extend. The electrode pairs a and B are mounted in an electroporation housing 12 which is slidably engaged with an indicator or gauge 11. As the electroporation wand housing 12 moves along the gauge 11, it alternately extends and retracts the electrode pairs a and B. The device indicator or gauge 11 may provide an indication of the extension length of the electrode pairs a and B. The electroporation device may further include an electrical connector 14 to electrically connect each of the first a and second B electrode pairs to a power supply 18, such as a pulse generator. The electrical connector includes four or more wires depending on the number of the electroporation electrodes for transmitting an electrical signal from the power supply to each of the electroporation electrodes. These signals may include the pin voltage set point, pulse width, pulse shape, number of pulses, and switching sequence. As described above, and as will be appreciated by those skilled in the art and described more fully below, the EPE may also function as an EME, in which case the generator may be capable of supplying both high voltage EP pulses and low voltage EIS interrogation signals, or a second low voltage power supply used with an appropriate switching mechanism to allow the delivery of higher voltage EP signals, and then lower voltage EIS signals.

In some embodiments of the invention, one or more of the EPEs may be a non-penetrating electrode, which may have an open distal end for administering the treatment portion to the tissue, as illustrated in fig. 7A and 11. The non-penetrating electrode may be any suitably shaped conductor, such as a button or plate to contact surface tissue. The injectors may be disposed in spaced relation to one another and in intimate contact with a surface region of tissue of the body. The portion of the non-penetrating electrode in contact with the tissue surface is electrically conductive and is electrically connected to a power supply, such as power supply 18, by an electrical connector, such as electrical connector 14, such that EP is achieved by completing an electrical circuit between the conductive distal ends of the non-penetrating EPEs to deliver current through the tissue region.

The EPE may be formed of a conductive material, but an optional insulating coating may be used as discussed herein. The EPE can be made of any conductive material capable of delivering the large instantaneous current density associated with the applied high voltage pulse, including but not limited to: certain metals and their oxides, including gold, platinum, palladium, silicon, aluminum; a metal oxide electrode comprising platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo) ₂ O ₆ ) Tungsten oxide (WO) ₃ ) And ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, and carbon paste). Preferably, the electrode comprises AgCl,Cobalt-chromium, titanium, stainless steel, platinum, gold, or metals plated with gold or platinum having high conductivity.

In some embodiments, for example when using a non-penetrating electrode pair, the distal end of the electrode is exposed for the generation of an electric field, but its proximal end may be coated with a non-conductive substance in order to limit the application of an electric field to only the distal end of the electrode adjacent to the tissue.

In some embodiments, an EP electrode configured for insertion may be similarly coated with an insulating material such that the electric field is generated using the distal end of the electrode and not along the length of the electrode, e.g., to allow EP to be "deeper" in tissue rather than in "shallow" regions.

In some embodiments, the intervening EP electrodes may have alternating regions of insulating material and bare electrode, as generally depicted in fig. 4A and 4B. In this embodiment, the electrodes may be coated with the same pattern, resulting in a more uniform electric field, or coated with different patterns, resulting in an asymmetric electric field. Similarly, the electrodes may have the same length or different lengths for all electrode configurations herein.

The pulsed electric field generated by these partially insulated electroporation electrodes is concentrated during treatment primarily in the regions between and near the exposed tip portions at the distal ends of the electrodes, and is small in the regions between and near the insulated portions. A partially insulated needle array may be used to limit electroporation in a target area having a tumor and to significantly shield skin and tissue beyond the target area from the electroporation process. This provides protection for healthy skin and tissue, which may be at risk due to undesirable or even adverse effects when some treatment portions are injected into healthy surface tissue above the target area.

Fig. 7A, 7B, 7C, and 7D depict different EPE configurations, but only a single pair of electrodes for simplicity. Fig. 7A depicts a set of non-penetrating solid EPEs applied topically to the surface of the skin. Additional sets of EPEs are not shown, but are included. FIG. 7B depicts a set of solid EPEs penetrating into tissue; in this embodiment, the tip of each EPE is substantially pointed to facilitate insertion into tissue, such as a solid needle tip. In this embodiment, the electric field region is "deeper" in the tissue, e.g., below the surface. This results in a three-dimensional electric field along the length and radial dimension between the electrodes. In general, these penetrating EPEs can range from about 1 to about 20mm, depending on the geometry and physiology of the tissue to be treated. Note that this measurement is the depth of insertion and not the total length of the electrode; there will generally be a portion of the electrode extending up from the point of contact with the tissue and into the wand housing for attachment to the appropriate circuitry, holding the electrode in the correct spatial configuration, and so forth. In fig. 7C, the penetrating solid EPE is coated with an insulating (non-conductive) material such that only the distal portion of the electrode is exposed. In the embodiment of fig. 7A, 7B and 7C, the TM delivery system will generally be a needle (not shown) that is inserted less deeply into the EP location between the EPEs. In fig. 7D, the penetrating EPE is hollow, with a lumen for TM delivery and a sharp open tip connected to the lumen. On the left, the penetrating electrode has a portion along the axis that is coated with an insulating material. As will be appreciated by those skilled in the art, when the capacitance measurement is complete, the EPE may additionally serve as an Electrical Measurement Electrode (EME) or there may be a separate set of EMEs, as generally depicted in fig. 6.

Figures 8A, 8B, 8C and 8D depict components of an EP apparatus of the present invention (which all rely on cylindrical needles, but other geometries may be used; and only a single pair of EP electrodes is depicted). Fig. 8A and 8B depict a set of EPEs (a second set not shown) with a TM delivery (TMD) system. Fig. 8A shows an EPE and TM delivery system inserted into tissue, where the TMD hollow needs to have an open end, a lumen is used to deliver the TM, and the TM is being delivered in a profiled manner. Fig. 8B shows the underside of the device, which may be in the distal end of the rod. Alternatively, as shown in fig. 8C, the TMD system may include a standard syringe that is manually inserted by the administering physician during the procedure. In this embodiment, the syringe may have an optional needle stop to physically prevent deeper penetration at a depth related to the depth of the electric field region. Fig. 8D depicts a TM delivery needle having multiple openings to deliver a TM. This may be useful when delivering larger biomolecules such as plasmids and antibodies, as larger molecules (which are otherwise generally charged) generally diffuse more slowly in tissues than other molecules. Having multiple delivery gene sites within the EP site can therefore be used to allow a higher percentage of cells in a region to take up TM. Fig. 8D depicts three openings or ports, but any number may be used. Additionally, fig. 8D depicts an opening on a "side" of the needle, but the opening may be located on any portion of the outer surface of the needle, forming a spiral or other shape.

In some embodiments, the electroporation electrodes generally have a length so as to completely surround the tissue to be treated. In a preferred embodiment, all sets of electrodes (the "array" of electrodes) have the same length within the array, but the use of different length electrodes may result in a modified and asymmetric electric field in some instances.

In many embodiments, the width and cross-sectional shape of the electrode for insertion is configured to minimize pain. Thus, the width of the electrodes may be from 0.05 to 1 to 2mm, and may depend on when the electrodes are also used to deliver the TM. Generally, when the electrodes are hollow and used for TM delivery they are generally larger to accommodate the lumen for TM delivery.

In addition, the electrodes and the wand housing are preferably made of a material that can be sterilized and configured to similarly minimize microbial entrapment in the event that the electrode array and wand housing are to be reused. In some embodiments, at least the electrode array is disposable, and in some embodiments, the entire rod housing is also disposable.

In some embodiments of the invention, multiple sets of electrode pairs are used. That is, as depicted in the figure, two sets (two pairs, four electrodes) may be used, for example, with first and second pairs of electroporation electrodes. The first and second pairs of electroporation electrodes are offset from each other by a predetermined angle. For a set of two pairs of electrodes, the two pairs of electrodes are offset from each other by an angle of about 90 degrees, as illustrated in fig. 1, with 90 degrees being preferred in some embodiments. The electrodes may also be positioned at a distance of 1 to 10mm and define the outer perimeter of the electric field region.

In some embodiments, one or more of the electrodes may be a hollow needle for introducing the treatment portion as discussed below.

The tissue surrounding the EP electrode is sometimes referred to as the "burn-out zone". By "burn-out" region is meant a region occupied by tissue in close proximity to and/or in contact with each of the individual electrodes. It is called the "burn-out" zone because the cells are in direct contact with the electrodes, which are heated due to the high voltage signal from the power supply, and thus the cells are subject to damage due to overheating. However, by using the alternating pulse device of the present invention, damage to the cells in the burn-out zone can be minimized by reducing the heat and field by 50% (in the case of two sets of electrodes; more if more sets are used). In addition, since the electric field strength is focused/increased at the electrodes, higher voltages can cause EP-mediated cell death in a heat-independent manner.

Figure 5 is a diagram of a hardware architecture for use in generating pulsed electric fields for the electroporation electrode pairs a and B. The electroporation device may be based on a Digital Signal Processor (DSP), microprocessor, field Programmable Gate Array (FPGA), application specific integrated circuit, central Processing Unit (CPU), or any multi-purpose programmable device that accepts analog/digital data as input, processes the input according to instructions stored in memory, and provides an output as a result. The switching sequence routine for electrode pairs a and B is programmed and stored in memory. The data bus may be used to display and modify the pulse parameters. High voltage isolation would allow hardware to be used with a high voltage power supply when plugged into a PC. A low voltage power supply may be used to power all auxiliary circuitry, such as capacitive or impedance sensing electrodes, analog-to-digital converters, digital-to-analog converters, repeaters, DSPs, optical switches, and the like.

In some embodiments, as illustrated in fig. 5, the first and second pairs of electrodes may be further connected to a generator of electrical signals capable of supplying various waveforms for each respective pair of EPEs. The first pair of EPEs a may be supplied with a waveform having a predetermined phase difference from the waveform supplied to the second pair of electroporation electrodes B by the power supply. For example, the first and second pairs of EPEs may receive waveforms having a phase difference of 180 degrees, as illustrated by the rectangular electrode pair a and electrode pair B waveforms shown in fig. 13. As will be appreciated by those skilled in the art and described more fully below, when EIS, the generator or power supply is capable of delivering both high voltage EP pulses and low voltage interrogation signals, and if not, additional low voltage EIS power supplies are provided.

In some embodiments, a highly specialized medical grade fast switching high voltage/high current solid state repeater is used to switch a generator from a low voltage EIS mode supplying an interrogation signal for EIS to a high voltage EP mode supplying an EP pulse for EP using an optically coupled relay driver. Each relay driver may be connected between a high voltage power supply and a corresponding pair of electroporation electrodes. Each relay channel may be implemented in a push-pull configuration to ensure that stray charge is removed from each of the electrode pairs during an off event.

An electrical power supply having first and second waveform generators may be electrically connected to the solid state high voltage relay channel a and the relay channel B to control and output first and second electrical signals, wherein the first and second waveforms reach the respective electroporation electrodes a and B.

B. Therapy delivery system

In some embodiments, the EP device of the present invention may comprise a Therapeutic Moiety (TM) delivery system. The TM delivery system may be integrated into the EP device in the form of a central probe or channel for TM delivery. In some embodiments, as described above, the EPE may be formed as a hollow electrode, having an open distal end for delivery of the TM therethrough. By "therapeutic moiety" ("TM") herein is meant a portion of the EP suitable for treating diseased tissue that contains a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, interleukin, or other therapeutically active agent. The TM may be a small molecule drug, a nucleic acid (including those encoding a therapeutic target protein), or a biologically active protein (including polypeptides and peptides).

In some embodiments, TM is a drug; the drugs contemplated for use in the methods of the invention are generally chemotherapeutic agents having anti-tumor or cytotoxic effects. These drugs or agents include bleomycin, neocarzinostatin, suramin, doxorubicin, carboplatin, paclitaxel, mitomycin C and cisplatin. Other chemotherapeutic agents will be known to those skilled in the art (see, e.g., the Merck index). EP promotes the entry of bleomycin or other similar drugs into tumor cells by creating pores in the cell membrane.

In some embodiments, the TM is a nucleic acid. Generally, a TM that is a nucleic acid has two distinct functional types. In one embodiment, the nucleic acid encodes a protein for treating a disease; in other embodiments, the nucleic acid is a TM, e.g., when the nucleic acid is an siRNA or snRNA. By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein is meant at least two nucleosides covalently linked together. The nucleic acids of the invention will contain mainly phosphodiester bonds, but in some cases, nucleic acid Analogues that may have alternating backbones, including, for example, phosphoramides (Beaucage et al, tetrahedron 49 (10): 1925 (1993) and references therein, letsinger, J.Org.Chem.) -35 3800 (1970), sprinzl et al, european journal of biochemistry (Eur.J.biochem.) -81 (1977), letsinger et al, nucleic acid research (Nucl. Acids Res.) -3487 (1986), sawai et al, rapid chemistry (chem. Lett.) -805 (1984), letsinger et al, U.S. Pat. J.chem.J.) -110 (1984), nucleic acid Analogues of nucleic acids, and nucleic acid Analogues of nucleic acids of the aforementioned nucleotide sequences, journal of the national academy of sciences (proc.Natl.Acad.Sci.USA) 92; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; kiedrowshi et al, applied to International publication on the chemistry (Angew. Chem. Int. Ed. English) 30 (1991); letsinger et al, J.Chem.Soc. 110 (1988); letsinger et al, nucleotide and Nucleotide (Nucleoside & Nucleotide) 13 (1994); ASC conference 580, carbohydrate modification in Antisense studies (Carbohydrate modification in Antisense Research) 2 and 3, Y.S.G. huui and P.Dan Coje et al, modification of Carbohydrate in Antisens Research, sanchel 395, J.23, J.R. Pat. No. 25 & N.5 & N.J. Biochem & S. 23 (Biotechnology) and Biotech. Ribos [ Biotechnology ] 32, J.11 & R. J. Pat. 5 & S. J.11 & N. Biotech (Biotechnology) 500, biotechnology, 2 & R. 3, biotech, 2 & S. Pat. 3, biotech, biotechnology, and Biotech, 2 & S. Proc. Pat. 3. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al, reviews of chemical sciences (chem. Soc. Rev.) (1995) pp 169-176). Several nucleic acid analogs are described by Rawls in chemical and engineering News (C & E News) 1997, 6, 2, page 35. All of these references are hereby expressly incorporated by reference herein. These modifications of the ribose phosphate backbone can be done to increase the stability and half-life of these molecules in physiological environments, for example when the nucleic acid is siRNA or the like.

In some embodiments, the nucleic acid is DNA or RNA encoding a therapeutic biomolecule, including a protein, including an antibody.

In some embodiments, the nucleic acid encodes an interleukin, which can be used to stimulate the immune system of a patient and/or cause apoptosis or necrosis in nucleic acid-transformed cells. Suitable interleukins include, but are not limited to, IL-12.

In some embodiments, the nucleic acid encodes a chemotherapeutic antibody. In general in this embodiment, there are two nucleic acids electroporated into the tissue, one encoding the heavy chain and one encoding the light chain. In some cases, these may be in a single expression vector or two expression vectors may be used.

The term "antibody" is used generally. Antibodies suitable for use in the present invention may take a variety of forms as described herein, including conventional antibodies as well as antibody derivatives, fragments and mimetics as described below. Conventional antibody building blocks typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention relates to the IgG class, which has several subclasses, including but not limited to IgG1, igG2, igG3, and IgG4, wherein the former are particularly useful in many applications, particularly oncology. Thus, "isotype" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It is understood that therapeutic antibodies may also comprise hybrids of the same type and/or subclass.

The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as the "Fv domain" or "Fv region". In the variable region, each V domain of the heavy and light chains aggregates three loops to form an antigen binding site. Each loop is called a complementarity determining region (hereinafter referred to as "CDR"), in which the variation in amino acid sequence is most pronounced. "variable" refers to the fact that the sequences of certain segments of the variable region differ widely by antibody. The variability distribution within the variable region is not uniform. In contrast, the V region is composed of relatively invariant segments of 15-30 amino acids, called Framework Regions (FRs), separated by shorter, extremely variable regions (called "hypervariable regions") each 9-15 amino acids in length or longer.

In some embodiments, the antibody is a full length antibody. By "full length antibody" herein is meant a structure that constitutes the native biological form of the antibody, comprising variable and constant regions, optionally comprising one or more amino acid modifications as known in the art. Alternatively, the antibody may be of a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively. Specific antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) A dAb fragment consisting of a single variable (Ward et al, 1989 nature 341, incorporated by full reference), (v) isolated CDR regions, (vi) an F (ab') 2 fragment, comprising two bivalent fragments linking a Fab fragment, (vii) a single chain Fv molecule (scFv), wherein the VH and VL domains are linked by a peptide linker which allows association of the two domains to form an antigen binding site (Bird et al, 1988 nature 242-426, huston et al, 1988 journal of the american college of sciences 85 5879-5883, incorporated by full reference), (viii) a bispecific single chain Fv (WO 03/11161, hereby incorporated by reference), and (bifunctional antibodies "or" trifunctional antibodies "multivalent or multispecific fragments constructed by gene fusion (tomdlinson et al, 2000, enzymology Methods (" ethol.) -326/479 ", journal of japan 6448, incorporated by national academy of sciences 6404, 1993). Antibody fragments may be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking VH and VL domains (Reiter et al, 1996 "Nature biotechnology (Nature biotech.)" 14, incorporated by full reference).

As will also be understood by those skilled in the art, the nucleic acid TM can be incorporated into a plasmid and/or expression vector, comprising additional components, including but not limited to expression promoters.

In some embodiments, the delivery system may comprise a rod delivery system configured to deliver the TM to the electroporation site. The delivery system may include at least one injection probe defining a first lumen, and the injection probe may be cylindrical in shape and have a needle tip at a distal end of the injection probe. The needle tip may be hollow and have an open end for delivery of the TM to the electroporation site. In some embodiments, the TM is injected into the middle of the outer perimeter defined by the EPEs and electroporated into the cells in the electroporation site using any of the EP devices described herein.

It is understood that EP of tissue can be performed ex vivo, in vivo, or in vitro. EP can also be performed with single cells, e.g. single cell suspensions, in vitro or in vitro in cell culture.

The EP wand housing, such as wand housing 12, is grasped and the EPE is inserted into the tissue to the desired depth. Subsequently, a suitable generator or power supply as described herein is connected to the EPEs and an appropriate voltage is applied to each of the pair of EPEs. A suitable amount of a therapeutic moiety such as a gene or molecule of a suitable chemical or drug for tissue treatment is then injected into the tissue using the rod delivery system described above, followed by application of EP pulses.

In some embodiments, the delivery system may include at least one injection probe defining a first lumen, and the injection probe may be cylindrical in shape and have a needle tip at a distal end of the injection probe. The needle tip may be hollow and have an open end for delivering the treatment portion to the electroporation site. In some embodiments, the treatment portion is injected into the middle of the outer perimeter defined by the pair of electroporation electrodes a and B and electroporated into the cells in the electroporation site 110 using the EP apparatus of the present invention.

C. Electroporation method

Various embodiments of the present invention are directed to methods of electroporating cells in an electroporation site of tissue using an electroporation system, such as system 10 of the present invention (illustrated in fig. 9). Various embodiments of the present invention are directed to the use of focused pulse-plus-electroporation. By "Focused Pulse Addition (FPA) electroporation" herein is meant the application of short electric field pulses to an electric field region through at least first and second pairs of electroporation electrodes to create transient pores in the cell membrane without permanent damage to the cell. "electroporated cells" include those cells having transiently open pores in the cell membrane that are closed upon dissipation of charge on the cell membrane ("open pore cells"), as well as those cells that have undergone electroporation such that the cells now contain an exogenously added therapeutic moiety and have closed pores (e.g., are intact again).

By "pair of electroporation electrodes" herein is meant a pair of two electrodes which, when connected to a power supply, are configured to be oppositely charged. The method may include contacting the electroporation wand housing 12 to tissue such that an electric field region 100 is defined by the area encompassed by the pair of electroporation electrodes a and B, as illustrated in the figure. The first and second pairs of electroporation electrodes may be stationary or retractable within the electroporation wand housing 12. The electroporation wand housing 12 may further include a circuit board 16 having a plurality of sliding sockets through which the electroporation electrodes A and B slidably retract and extend. The electrode pairs a and B are mounted in an electroporation housing 12 which is slidably engaged with an indicator or gauge 11. As the electroporation wand housing 12 moves along the gauge 11, it alternately extends and retracts the electrode pairs a and B, as illustrated in fig. 2. The device indicator or gauge 11 may provide an indication of the extension length of the electrode pairs a and B. The electroporation system may further include an electrical connector 14 to electrically connect each of the first a and second B electrode pairs to a power supply 18, such as a pulse generator. The electrical connector includes four or more wires depending on the number of the electroporation electrodes for transmitting an electrical signal from the power supply to each of the electroporation electrodes. These signals may include the pin voltage set point, pulse width, pulse shape, number of pulses, and switching sequence. As will be appreciated by those skilled in the art and described more fully below, the EP electrode may also function as a CS or EIS electrode, in which case a second low voltage power supply is used with a suitable switching mechanism to allow delivery of a higher voltage EP signal, and then a lower voltage CS or EIS signal.

In some embodiments of the invention, one or more of the electroporating electrode pairs a and B may be a non-penetrating electrode, which may or may not have an open distal end for administering the treatment portion to the tissue, as illustrated in fig. 11. The non-penetrating electrode may be any suitably shaped conductor, such as a button or plate to contact surface tissue. The injectors may be disposed in spaced relation to one another and in intimate contact with a surface region of tissue of the body. The portion of the non-penetrating electrode in contact with the tissue surface is electrically conductive and is electrically connected to the power supply 18 by the electrical connector 14 such that electroporation is achieved by completing an electrical circuit between the conductive distal ends of the non-penetrating electroporation electrodes to deliver electrical current through the region of tissue.

In some embodiments, as illustrated in fig. 12, more than two pairs of electrodes may be used and the offset angle may be adjusted accordingly. The greater the number of electrodes, the more electroporation sites become focused the electric field across all electrode pairs. Furthermore, the large number of electrode pairs allows for shorter pulses from each electrode pair, thereby substantially reducing or even eliminating cell death and burns around the electrodes in the "burn zone".

The tissue surrounding the EP electrode is sometimes referred to as the "burn-out zone". By "burn-out" region is meant a region occupied by tissue in close proximity to and/or in contact with each of the individual electrodes. This is called the "burn-out" zone because the cells are in direct contact with the electrodes, which are heated due to the high voltage signal from the power supply, and thus the cells are subject to damage due to overheating. However, by using the alternating pulse system of the present invention, damage to the cells in the burn-out zone can be minimized by reducing the heat and field by 50% (in the case of two sets of electrodes; more if more sets are used). In addition, since the electric field strength is focused/increased at the electrodes, higher voltages can cause EP-mediated cell death in a heat-independent manner.

In some embodiments, as illustrated in fig. 13, the first and second pairs of electrodes may be further connected to an EP power (EPP) supply capable of supplying electrical signals of various waveforms to each respective pair of electroporation electrodes. That is, the power supply may be a high voltage power supply suitable for waveform generation. The first pair of electroporation electrodes a may be supplied with a waveform having a predetermined phase difference from the waveform supplied to the second pair of electroporation electrodes B by the power supply. For example, the first and second pairs of electroporation electrodes may receive waveforms from the first and second waveform generators, respectively, of the power supply, the waveforms having a phase difference of 180 degrees, as illustrated by the rectangular electrode-pair a and electrode-pair B waveforms shown in fig. 13 and 14A and 14B. As will be appreciated by those skilled in the art and described more fully below, when capacitive sensing is complete, a low voltage power supply is optionally used.

The method can include contacting the electroporation wand housing 12 to tissue such that an electric field region 100 is defined by the area encompassed by the pair of electroporation electrodes a and B, as illustrated in fig. 10 and 14A and 14B. In some embodiments, the electroporation method may further include applying the generated first signal from the power supply to the first pair of electroporation electrodes a with a first waveform and applying the second signal from the power supply to the second pair of electroporation electrodes B with a second waveform, wherein the first waveform has a predetermined phase difference from the second waveform.

The electroporation system 10 sends a plurality of independent electrical signals to selected pairs of electrodes a and B during operation that cause electroporation in the cell membrane when contacting tissue. When the first and second electrode pairs a and B are in electrical contact with the tissue, the first electrical signal and the second electrical signal having the first frequency combine to produce a constant waveform having a frequency and amplitude sufficient to temporarily open the pores of the cells for treatment of a portion of the cells optionally introduced into the cells of the tissue without permanently damaging the cells and minimizing pain.

The nature of the tissue, the size of the selected tissue and its location may determine the nature of the electrical signal to be generated. It is desirable that the electric field be as homogeneous as possible and have the correct amplitude. Excessive electric field strength can lead to cell death, while low field strength can lead to inefficient electroporation of the cells, thus reducing the efficiency of delivering agents into the cells.

The method may further include applying a pulsed electric field to the electric field region 100 from the first pair of electroporation electrodes a, the pulsed electric field based on the first signal, wherein the pulsed electric field and each subsequent pulsed electric field of the first pair of electroporation electrodes a have a voltage and duration below a minimum threshold for electroporation. Then, another pulsed electric field is applied to the electric field region 100 from the second pair of electroporation electrodes B, the another pulsed electric field being based on the second signal, wherein the another pulsed electric field and each subsequent pulsed electric field of the second pair of electroporation electrodes B have a voltage and duration below a minimum threshold for electroporation.

According to the method of the present invention, the paths of the pulsed electric fields of the first and second pairs of electroporation electrodes a and B intersect at the electroporation site 110, and the application of each pulsed electric field of the first pair of electroporation electrodes to the electroporation site alternates with the application of each pulsed electric field of the second pair of electroporation electrodes to the electroporation site, in total, as a continuous pulsed electric field having a voltage and duration sufficient for applying electroporation to cells in the electroporation site, as illustrated in fig. 14A and 14B.

In another aspect, each application of a pulsed electric field of the first pair of electroporation electrodes to tissue adjacent to and outside of the electroporation site alternates with a resting period such that tissue adjacent to and outside of the electroporation site receives from the first pair of electroporation electrodes alternating on and off pulsed electric fields having a voltage and duration below a minimum threshold for electroporation. Wherein the application of each pulsed electric field of the second pair of electroporation electrodes to tissue adjacent to the second pair of electroporation electrodes and outside of the electroporation site alternates with a resting period such that tissue adjacent to the second pair of electroporation electrodes and outside of the electroporation site receives an alternating on and off pulsed electric field of the second pair of electroporation electrodes having a voltage and duration below a minimum threshold for electroporation as illustrated in figures 14A and 14B.

Thus, the electroporation method of the present invention yields the advantage that healthy cells outside the electroporation site but within the electric field region 100 are subjected to the electric pulse for only half the duration of those cells in the electroporation site and are insufficient for electroporation, which cells maintain minimal to no permanent damage. Furthermore, since cells outside the electroporation site 110 but within the electric field zone 100 are subjected to only short pulses, this minimizes the extent of damage to cells in the "burn-out" zone immediately adjacent to the electroporation electrodes.

The nature of the tissue, the size of the selected tissue and its location may determine the nature of the electrical signal to be generated. It is desirable that the electric field be as homogeneous as possible and have the correct amplitude. Excessive electric field strength can lead to cell death, while low field strength can lead to inefficient electroporation of the cells, thus reducing the efficiency of delivering the agent into the cells.

In some embodiments, the pulsed electric field is selected from the group consisting of: square wave pulses, exponential wave pulses, finite duration unipolar vibration wave forms, and finite duration bipolar vibration wave forms.

According to various methods of the present invention, as illustrated in fig. 10, the paths of the pulsed electric fields of the first and second pairs of EPEs a and B intersect at the electroporation site 110, and the application of each pulsed electric field of the first pair of EPEs to the electroporation site 110 alternates with the application of each pulsed electric field of the second pair of EPEs to the electroporation site 110, in total, into a continuous pulsed electric field having a voltage and duration sufficient for electroporation of cells in the electroporation site 110, as illustrated in fig. 14A and 14B.

Preferred apparatus embodiments

(i) Integrated EP device

Accordingly, the present invention provides devices and methods for improved delivery of a therapeutic moiety to cells in a tissue of a patient. An integrated device for improved delivery of a treatment portion to cells in a treatment region of a tissue is described. The device includes at least a helical probe 1702 having an inner surface, which in some embodiments may be a central probe, as illustrated in fig. 17A, and in some embodiments may include at least one additional probe 1702, as illustrated in fig. 18A and 18B. Each of the central probe and the additional probes can define one or more central lumens 1704 (e.g., a first central lumen).

The first central lumen 1704 extends from the proximal end 1706 to the distal end 1708 of the central probe 1702. In some embodiments, the proximal end of the central probe may be formed of or coated with a non-conductive material to prevent or reduce the generation of an electric field at the proximal end. The proximal end 1706 of the central probe 1702 may define an opening to fluidly connect the central lumen with a lumen of an injector through which a therapeutic agent may be delivered to the central probe 1702. In some embodiments, the distal end 1708 of the central probe also defines an opening for delivering the treatment portion into tissue. Alternatively, the distal end 1708 may be closed, as illustrated in fig. 22A to 22C. One or more portions of the distal end of the central probe 1702 may have a shape configured to pierce tissue.

The central lumen 1704, or portion of the central lumen, includes a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel 1734 for delivering the TM to the tissue via an injection port positioned on the central probe 1702. For example, a portion of the central probe 1702 may include one or more jet ports 1710 positioned along the geometry, e.g., as illustrated in fig. 17B.

In some embodiments, the central probe 1702 may be at least partially housed in the applicator 1712. The applicator may include a distal end through which portions of the central probe extend out of the applicator 1712 to contact tissue and retract into the applicator 1712. For example, the EP device may include an actuator to advance the central probe 1702 toward and through the distal end of the applicator and through the tissue 1714.

One or more diameters defined along the interior and/or exterior surface of the central probe may be adjustable to vary the distribution and volume of the delivered treatment portions. Similarly, the helical diameter and pitch of the central probe are adjustable to vary the distribution and volume of the delivered therapeutic moieties.

In some embodiments, the EP device may also include an electrical connector 1716 for electrically coupling or connecting the central probe 1702 to a source of electrical power. The electrical connector may be contained or housed in the handle 1718.

In some embodiments, the EP device may also include an electroporation system comprising two or more oppositely charged electroporation electrodes (EPEs) 1720. The two or more electrodes are configured to be positioned such that they substantially surround the treatment region 1722 during treatment. Electrode 1720 is adapted to extend from a proximal end to a distal end. One or more of the tips 1724 of the distal ends of the electrodes contain a needle shape for piercing tissue. The electrodes may be coupled to an electrode power supply (e.g., generator a illustrated in fig. 16) such that the electrodes receive one or more electrical waveforms from the power supply for supplying electrical pulses 1730 to generate a pulsed electric field sufficient for electroporation of the treatment region 1722 as illustrated in fig. 14A.

Similar to the central probe, the electrodes may be housed in an applicator. Electrodes 1720 may be positioned around the central probe 1702 and configured to be deployed from the applicator 1712 to the treatment region 1722. For example, the electrodes may be advanced from the applicator toward the treatment site and retracted into the applicator.

In some embodiments, the advancement and retraction of the electrodes may be powered by a power supply interface contained in the handle 1718. For example, the power supply interface may supply power to actuate the extension and retraction of the central probe 1702 and EPE 1720.

In other embodiments, as illustrated in FIG. 19, the central probe 1702 may include electrodes positioned on the spiral geometry of the central probe 1702. In these embodiments, the electrodes may be integrally formed with the central probe 1702 or may be removably disposed thereon. The electrodes on the central probe 1702 can be used in combination with the electrodes 1720 to produce a desired electric field configuration.

In other embodiments, as illustrated in fig. 20A and 20B, the central probe may be an electrode probe 1750 that is connected to an electrode power supply, such as generator a of fig. 16, such that an electric field is generated between the central probe 1750 and the EPE 1720 to facilitate electroporation. In some embodiments, as illustrated in fig. 20A, the central probe 1750 may include helical blades for creating channels and for better anchoring of the central probe 1750 in tissue. Fig. 20B is a schematic view of a center 1750 surrounded by a plurality of electrodes 1720, and fig. 20C is a bottom view illustration of fig. 20B.

The one or more center probes may include a second helical probe defined similarly to center probe 1720 herein. In this case, the second probe supplies a second channel for delivery of the treatment portion to the tissue.

In some embodiments, as illustrated in fig. 22A, one or more distal electrodes 1752 may be positioned at the distal end of the applicator 1712. These distal electrodes 1752 may be configured to generate an electric field with one or more portions of the central probe 1702. The one or more distal electrodes may be configured based on a loop configuration, a straight wire configuration, a helical wire configuration, or a collapsible loop configuration. As mentioned, the device may comprise ejection ports located on one or more portions of the central probe. The distal electrode may be configured to be positioned outside of the tissue to externally generate an electric field experienced by the tissue. Alternatively, the distal electrode 1752 can be configured to be positioned below the surface of the tissue. In some embodiments, as illustrated in fig. 21, the distal electrode 1754 may be formed based on a helical wire configuration such that the helical wire electrode is positioned below the surface of the tissue. The helix of the central probe 1702 and the helix of the distal electrode 1754 may be wound in opposite directions, as illustrated in fig. 21.

In some embodiments, the electrodes described herein (including distal electrodes) are housed in the applicator 1712 around the central probe 1702 such that they can be deployed accordingly from the applicator 1712 to substantially encompass the treatment region.

In other embodiments, one of the probes does not comprise a helical geometry. For example, one of the probes is a straight probe having open proximal and distal ends for delivery of the treatment portion to the tissue. The vertical axis of the straight probe is coaxially aligned with the central axis of the diameter of the central probe. The straight probe may be configured to generate an electric field with a portion of the central probe. A central probe having a helical geometry may be configured to transmit acoustic energy received from an acoustic horn mounted to the distal end of the applicator.

a. Sensor system

In some embodiments, the invention may include a sensor system. As will be appreciated by those skilled in the art, successful electroporation occurs when the cell membrane breaks, resulting in a change in capacitance. When subjected to an electric field, the cells generally act as capacitors. When the electric field is applied for a sufficiently long period (depending on the cell properties, health, size, etc.), charge accumulates at the cell membrane until it reaches a certain threshold and causes disruption of the membrane integrity. The capacitive sensor system may be a low voltage interrogation or stimulation circuit that may include a pair of capacitive sensing electrodes powered by a low voltage power source, a voltage sensor, a current sensor, and an electronic signal processing device to process the voltage and current to determine an average capacitance of cells in the region.

In these embodiments, the sensor system is used to perform impedance measurements of cell membranes of tissue and includes a pair of capacitive sensing electrodes (e.g., electrodes 1720) powered by a low voltage power supply (e.g., generator a illustrated in fig. 16). The sensor system may further include a voltage sensor (integrated into the electrodes 1720, 1752, and/or 1754) configured to sense a voltage or voltage drop across a cell membrane. In addition, the sensor system may include a current sensor (integrated into the electrode 1720) configured to sense current across the cell membrane and an electronic signal processing device, such as the controller 1505 illustrated in fig. 15. An electronic signal processing device (e.g., controller 1505) processes the voltage drop and current across the cell membrane to determine the impedance of the cell membrane.

In some embodiments, the above-described method for sensing impedance (EIS) may include applying a waveform, such as a phase-locked loop, square wave pulse, high frequency pulse, chirp, or the like, as described more fully below. When exposed to an electric field, the cell membrane acts as a capacitor. Capacitance may be measured based on charge redistribution in the cells in response to low frequency electric field excitation of the low voltage interrogation circuit, and impedance measurements may be derived from the capacitance. Capacitance can be measured before, during, and after application of the electroporation electric field to determine cell conditions, including but not limited to cell health, placement of electrodes relative to the cell for optimal electroporation, and most importantly a time constant, which can be used to determine the pulse width of the electric field to be applied to the cells in the electric field region. In general, it takes five time constant periods to charge the capacitor to its maximum value, i.e., just before electroporation occurs, so the pulse width of the initial electroporation electric field pulse can be set to 5 time constants. This pulse width is insufficient to cause electroporation in cells outside the electroporation positions, as described above, but sufficient to cause electroporation in cells of tissue in electroporation positions that are subjected to the additive effect of electric fields from all of the collective electroporation electrodes being applied as one continuous electric field. The capacitance measurement may be repeated after the first electroporation electric field has been applied, and the percentage decrease in capacitance may be calculated and compared to a predetermined value to determine whether the cells in the electroporation site have been sufficiently electroporated. If not, the pulse width may be adjusted for the next set of electroporation pulse electric fields based on the calculated percent drop in capacitance until it is determined that sufficient electroporation has occurred in the electroporation location.

An electronic signal processing device (e.g., controller 1505) may fit the tissue impedance data to an equivalent circuit model, i.e., the CPE-based tissue model described above, in order to predict the next optimal pulse parameters. As described above, the electrical impedance is the sum of resistive and capacitive elements over a range of frequencies, so to quantify each of these parameters, tissue impedance data can be fitted to a CPE-based tissue model. Thus, capacitance measurements made between pulses by

electrodes

1720, 1752, and 1754 with integrated sensors allow for adjustment of electrical conditions, such as pulse width, based on time constants associated with cell membrane capacitance, and the electroporation process can be stopped when a desired decrease in capacitance or membrane integrity is reached. It is hypothesized that real-time monitoring of the electrical properties of the tissue will enable feedback control of EP parameters and result in optimal transfection in heterogeneous tumors. Using EIS feedback will allow (1) real-time adjustment of delivery parameters, (2) delivery of only the pulses necessary to produce a therapeutic response, and (3) reduction of overall EP-mediated tissue damage.

b. Therapeutic moiety delivery method

Various embodiments of the present invention are directed to methods for delivering a treatment portion to cells in a treatment zone of tissue using a delivery device integrated into an EP device having electrodes, such as central probe 1702 having ejection ports 1710 or central probe 1750 having ejection ports 1751. In some embodiments, a method for delivering a treatment portion to a treatment region of tissue may include providing an EP device having a central probe as a delivery device. In some embodiments, the delivery device includes a

central probe

1702, 1750 having an inner surface that defines at least a first central lumen and extends from a proximal end to a distal end of the

central probe

1702, 1750. In some embodiments, at least a portion of the delivery device 1702 has a helical geometry configured to enhance anchoring of the delivery device 1702 in tissue and create a channel for delivery of the treatment portion to the tissue. The portion of the central probe delivery device 1702 may have multiple ejection ports positioned along a helical geometry, where the proximal end of the central probe delivery device 1702 is open and the central lumen to which the therapeutic agent is delivered is fluidly connected to the cells or tissue. The distal end of the central probe delivery device 1702 is open to define an opening/ejection port 1710 for delivering the treatment portion into tissue, and has a shape configured to pierce tissue.

In other embodiments, the central probe delivery device 1750 illustrated in fig. 20A-20C has a straight tubular shape that includes blades 1753 having a helical geometry configured to enhance anchoring of the delivery device 1750 in tissue and create channels for delivery of the therapeutic moiety to the tissue. At least a portion of the central probe delivery device 1750 may have at least one injection port 1751 positioned thereon to fluidly connect a central lumen to which a therapeutic agent is delivered to a cell or tissue.

The method further includes contacting the central probe to diseased cells in a treatment area of the tissue, actuating and extending the central

probe delivery device

1702, 1750 in an axial direction from the applicator, piercing the tissue with at least a portion of the central

probe delivery device

1702, 1750 and creating an opening through which at least a portion of the central probe enters the tissue to create a fluid channel for delivery of the treatment portion to the tissue, injecting the treatment portion into the central lumen and delivering the treatment portion to the tissue through the at least one ejection port 1751 and the open distal end of the central probe.

In some embodiments, the method further comprises providing an electroporation system or device comprising at least two oppositely charged electroporation electrodes, such as electrode 1720 positioned configured to surround a region of tissue, wherein the electroporation electrodes are adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, and the electroporation electrodes are adapted to be coupled to a source of electrical power. The method further includes contacting a region of tissue with an electroporation electrode, delivering an electrical pulse from the electrical power source to the electrode, and applying a pulsed electric field sufficient for electroporation to the region from the electroporation electrode.

In some embodiments, a method for delivering a treatment portion to a treatment area of tissue includes providing a device for delivering the treatment portion to the treatment area of tissue. The method further includes contacting the central probe (e.g., 1702) and the distal electrode 1752 to diseased cells in a treatment region of tissue, actuating and extending the central probe 1702 and the distal electrode 1752 from the applicator in an axial direction, piercing the tissue with the distal electrode 1752 and with at least a portion of the central probe 1702 and creating an opening through which at least a portion of the central probe enters the tissue to create a fluid channel 1734 for delivery of the treatment portion to the tissue, injecting the treatment portion into the central lumen 1704 and delivering the treatment portion to the tissue through at least one of the ejection port and the open distal end of the central probe, delivering electrical pulses from the electrical power source to the distal electrode and the central probe, applying a pulsed electrical field from the distal electrode and the central probe sufficient for electroporation to the region, and retracting the distal electrode 1752 and the central probe 1702 from the tissue.

In some embodiments, as described above, the method for delivering a treatment portion to a region may further include coupling the EPE to a power source, contacting the region of tissue with the EPE, delivering electrical pulses from the power source to the electrodes, and applying a pulsed electric field sufficient for electroporation to the region of tissue from the EPE. Embodiments of the invention add the advantage of opening the pores of the cells to the delivery method of the invention, thereby allowing the cells to absorb a larger volume of the therapeutic moiety and produce better therapeutic results.

In some embodiments, as described above, the method for delivering a therapeutic moiety to a zone may further include providing a capacitive sensing system and method in conjunction with an electroporation system and method for optimization of electroporation parameters, as described in more detail below. When exposed to a low frequency, low intensity electric field, the cells generally behave as an insulating structure surrounded by ion clouds that compensate for the fixed charges present in the thin film. The electric field polarizes the ion cloud and creates an electric dipole, which causes the cell to act as a capacitor. Healthy cells act as stronger capacitors than dead or diseased cells with damaged thin film structures, thereby resulting in stronger capacitive coupling between the cells and the capacitive sensing electrodes. Thus, these properties can be used as an indication of the integrity of the thin film of the cell, which in turn will yield a determination of the extent of electroporation of the cell.

The method for delivering a therapeutic moiety to a region of tissue may further comprise sensing cell membrane capacitance of the tissue in order to optimize the electroporation process.

In some embodiments, the methods of the present invention may comprise contacting tissue in the region of tissue with the pair of capacitive sensing electrodes (e.g., 1720). A low voltage power supply (e.g., generator a) electrically connected to the capacitive sensing electrode is used to apply a low voltage interrogation signal to the capacitive sensing electrode. Methods for sensing capacitance may include, but are not limited to, waveforms such as phase locked loops, square wave pulses, high frequency pulses, and chirps. Voltage and current sensors are used to sense the voltage drop and current flowing through the circuit, and these parameters can then be processed by an electronic signal processing device to determine the average capacitance of all cells in the measurement area. As described above, the measured capacitance is an indicator of the health of the cell and is used to determine how long an electrical pulse is applied in order to disrupt the cell membrane and provide conditions sufficient for electroporation.

Fig. 23A, 23B and 23C, 23D, 23E and 23F illustrate various EP devices with a central probe having a helical geometry as described above according to the present invention.

(ii) EP device based on trocar device

The present invention thus provides a system for improved EP of a cavity not readily accessible within the body. Fig. 24A, 24B and 24C illustrate a trocar-based direct rod applicator EP system according to the present invention. The trocar-based direct stick applicator EP system device design allows for immunotherapeutic agent gene delivery to tumors inaccessible to the skin electroporation device. Examples of such cases are the case where the lung, liver, breast or any tumor is more than 10cm below the skin. The EP system provides the advantage of improved co-localization of DNA and electric fields for efficient gene delivery and reduction of user-induced changes.

In some embodiments, a system for Electroporation (EP) of cells in tissue of a subject may comprise a trocar comprising a cannula 2402 and an obturator 2404, and an EP device 2406 slidably mounted and retracted within the cannula 2402 to access the cells or tissue. In some embodiments, the cannula 2402 extends from a proximal end to an open distal end 2408 and defines a first lumen configured to receive an obturator 2404, and the obturator extends from a proximal end 2410 to a distal end 2412. The proximal end of the obturator may include a handle mounted thereon, and the distal end of the obturator may include a blade or sharp tip 2414 configured to pierce through the skin, penetrate into the body cavity, and form a pathway through which the cannula 2402 may be inserted at least partially into the body cavity. In some embodiments, the obturator 2404 is configured to be slidable within the first lumen, and the distal end 2412 of the obturator is configured to extend out of the first lumen through the open distal end of the 2402 cannula.

In some embodiments, EP device 2406 includes an anchor 2418 extending proximally to a distal end 2420, at least two oppositely charged electrodes 2422, a central probe 2424 (which may be configured in the same manner as helical probe 1702 with open distal end 1708) retractably disposed at the distal end 2420 of the anchor. In some embodiments, the at least two oppositely charged electrodes 2422 are retractably disposed at the distal end 2420 of the anchor 2418 and are configured to be positioned surrounding a region of a target cell, such as region 1722 of fig. 17A. In some embodiments, a measurement device is coupled to the electrode. The electrodes are adapted to be coupled to a generator, such as generator a of fig. 16, receive at least one electrical waveform from the generator, and supply at least one of an excitation signal and an EP pulse to tissue in the region. The central probe may have an inner surface defining at least a central lumen and extend from the distal end of the anchor. At least a portion of the central probe 2424 can have a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the treatment portion to tissue in a similar manner as described with respect to 17A-22C of the present invention. A distal end 2428 of the central probe 2424 may be open to define an opening for delivery of the treatment portion to tissue and may have a shape configured to pierce tissue. When the central probe is deployed, the anchor 2418 may be coupled to the proximal end of the central probe 2424.

In some embodiments, each helix of the central probe 2424 can range from 1mm diameter to 6mm diameter, typically from about 1mm to 3mm, more typically from 1.2mm to 2.3mm, and in some cases approximately 1.5mm. In some embodiments, the electrodes may be spaced apart from 2mm to 10mm, more typically from 2mm to 5mm, and in some cases approximately 2mm. In some embodiments, the length of the central probe and the length of the electrodes may vary from 5mm to 15mm, more typically 7mm to 10mm, and in some cases approximately 8mm. Although recited in terms of certain ranges, it is to be understood that all ranges from the lowest of the lower limits to the highest of the upper limits are included in these full ranges or any specifically recited range, including all intermediate ranges or specific measurements.

In some embodiments, the anchor is configured to be engaged by a 12ga biopsy needle to achieve a depth of 10cm via the biopsy needle. In this way, the EP device can be anchored to a soft tumor for increased dispersion of DNA. The EP apparatus of the present invention provides the advantage that only 87V needs to be applied across a 2.5mm spacing between the electrodes in order to achieve a field strength of 350V/cm. Electric field strengths of this magnitude have been associated with significant enhancements in TM delivery.

In some embodiments, the blade or sharp tip 2414 of obturator 2404 is configured to extend out of the cannula 2402 through an opening at the distal end 2408 of the cannula 2402. The EP device electrode 2422 may be adapted to extend from a proximal end to a distal end, a tip of the distal end may have a needle shape configured to pierce tissue, and the electrode 2422 may be adapted to be coupled to a generator, receive at least one electrical waveform from the generator, and supply at least one of an excitation signal and an EP pulse to a region of a target cell.

Various embodiments of the present invention are directed to providing methods for delivering therapeutic moieties to cells in tissue and EP of the cells using the EP apparatus of the previous embodiments. In some embodiments, the method includes (i) inserting the central probe into the anchor device, (ii) deploying the electrode, (iii) partially withdrawing the central probe, and (iv) injecting the treatment portion into a lumen of the central probe for delivery of the treatment portion to the tissue through a distal end thereof. In some embodiments, the method may further comprise (v) withdrawing the central probe; and (vi) applying electrical pulses from generator 1530 to the electrodes for electroporation; and (vii) a removal device.

(iii) Device for improved therapeutic agent delivery

Accordingly, the present invention provides devices and methods for improved delivery of a therapeutic moiety to cells in a tissue of a patient. Fig. 25 to 33 illustrate EP devices for improved therapeutic agent delivery according to the present invention.

As depicted in fig. 27, the EP device includes a central probe 2710 having an inner surface 2712 defining a first central lumen 2715 through which at least one access line 2720 may extend out of the central probe 2710 and may be retracted into the first central lumen 2715. The central probe 2710 further includes an exit port 2730 that fluidly connects the first central lumen 2715 to the exterior of the central probe 2710 and through which the injected treatment portions flow from the first central lumen 2715 into the channels in the cells. The EP device also includes a ramp 2760 integrally formed or coupled with the inner surface of the central probe to guide the score line 2720 out of the central probe 2710 to the diseased tissue or cells.

In some embodiments, the central probe 2710 has a closed distal end and a proximal lumen. The distal tip of the probe 2710 is fashioned in any shape designed to pierce tissue. Proximal to the distal tip, an exit port 2730 exposes the first central lumen 2715 to the exterior of the central probe/needle 2710. The access wire 2720, also having a piercing feature molded into its distal tip, is sized so that it is slidable within the first central lumen 2715 and exits through the exit port 2730. The access line 2720 is adapted to advance into the tissue of the tumor and create a channel through the tissue that serves as a fluid path for a treatment portion injected at a later time in the procedure. The lane line 2720 is directed outward by a ramp 2760 in the central probe 2710, as illustrated in fig. 25-33. The lane wire 2720 is adapted to retract into the central probe 2710 and the EP device can be rotated to a new orientation. The access wire 2720 may be repeatedly advanced into the cells to create additional channels for therapeutic moiety delivery. The channel created by the access line 2720 enhances retention of the injected treatment portion in the tissue and allows for injection of a larger volume than would be possible from a typical needle/syringe of similar size.

In some embodiments, the EP device may further include a handle that automates the extension, retraction, and rotation of the central probe/needle 2710 and the access line 2720 to facilitate sufficient depth penetration.

In other embodiments, such as the catheter or endoscope-based EP device of fig. 25 and 26, the EP device will contain a similar central probe/needle 2710 as described in the main embodiment.

In other embodiments, such as the EP device of fig. 30, the EP device will contain a similar central probe/needle 2710 as described in the primary embodiment. This embodiment will have multiple exit ports 2730 through which multiple lane lines may exit the device simultaneously.

In some other embodiments as illustrated in fig. 29, the access wire 2720 comprises a wire having a cutting blade 2773 molded in the distal end. The blade may be extended into the tumor and subsequently rotated so as to create a disc-like incision in the tumor to form a channel through which the treatment moiety is delivered to the cells.

In yet another embodiment, as illustrated in fig. 31, the central probe has an open distal end similar to a typical syringe/needle. The steering wire 2720 may be formed of a shape memory alloy, such as a superelastic material (e.g., nitinol), to allow the curve to be heat set into the wire (sometimes referred to as "shape memory"). When the access wire is in the central probe 2710, the wire is elastically straightened. Upon withdrawal from the central probe/needle, the access line 2720 is allowed to return to its curved shape, as illustrated in fig. 31, thus creating a channel extending outward from the device.

In yet another embodiment, as illustrated in fig. 30, the EP device includes an injection probe 2745 having an injection needle at its distal end for injecting a therapeutic agent, and a central probe 2755 having a separate lumen for guiding the access line 2720. The inner surface 2712 of the central probe 2755 can contain a ramp 2760, such as illustrated in fig. 25, 29, and 30, for guiding the access line 2720 outward from the central probe 2755 into the interior of the tumor. The two components creating the two lumens are joined side by a method suitable for the materials of construction thereof. For example, if the two lumens are made of metal, they may be spot welded together, and if the two lumens are made of a material such as hard plastic, they may be ultrasonically welded together. Alternatively, a single component of integrally formed multi-lumen tubing may be utilized to accomplish the same purpose. This configuration creates advantages in treatments where a larger infusion lumen is required for delivery in the treatment area.

Referring to fig. 27, an apparatus for improved delivery of a therapeutic moiety to cells in a tissue according to some embodiments of the present invention further comprises: an electrical connector 2770 that electrically connects the central probe 2710 and the access line 2720 to a power source 2780; a small aperture connector 2795 configured to connect the central probe 2710 to a syringe for delivery of the treatment portion; and a handle 2790 that houses an electrical connector 2770 and is coupled to the central probe 2710 and to the proximal end of the access line 2720 to facilitate the penetration depth of the central probe at the distal end of the access line 2710.

As illustrated in fig. 26, 27, 28 and 29, the central probe 2710 extends vertically outward from its proximal end to a closed distal end, and is configured with a needle shape at the distal end to provide initial penetration into the tumor/tissue. The inner surface 2712 of the central probe 2710 defines a first central lumen 2715 and is configured with a ramp 2760 that guides a guidewire 2720 outward from the EP device. The first central lumen 2715 provides a path along which a therapeutic portion for infusion flows prior to delivery to diseased cells or tissue.

In some embodiments, an exit port 2730 through which the treatment portion is delivered to diseased cells or tissue is positioned on the side surface of the central probe 2710 at a predetermined distance from its distal end. Exit port 2730 fluidly connects the central lumen out of the central probe. The central probe 2710 may be formed of a low conductive material that is coated at the distal end with an insulating (non-conductive) material to avoid interference with the electric field that may optionally be applied using EPEs, in order to facilitate uptake of the therapeutic moiety by the cells. The central probe 2710 can measure from about 1mm to about 10mm, depending on the geometry and physiology of the tissue to be treated and how deep into the tissue the access line 2720 and EPE 2750 need to be inserted.

In some embodiments, the access line is positioned in the central lumen and is slidable within the central probe. The access line may have a proximal end positioned in the central probe and a distal end with a needle-like puncture configured to extend out of the central probe through the exit port 2730 to reach and penetrate the diseased cells and create a fluid channel through which the therapeutic portion may be delivered to the tissue. The steering wires may be formed at the distal end of a low conductive material or an insulating (non-conductive) material coated with a conductive material in order to avoid interference with the electric field that may optionally be applied using the EPE. The access line 2720 may measure from about 1mm to about 20mm, depending on the geometry and physiology of the tissue to be treated and how deep the access line 2720 and EPE2750 need to be inserted into the tissue.

As illustrated in fig. 29, the ramp may be integrally formed or coupled with the inner surface 2712 of the central probe and may be adapted to contact and guide the access line back out of the central probe to the outside of the central probe. The ramp 2760 may be formed or coupled to the inner surface of the central probe at a predetermined angle, which may or may not be adjusted based on the extension angle necessary to get the lane 2720 to the diseased cell.

Referring to fig. 27, to supply power to the EP delivery device, an electrical connector 2770 electrically connects the central probe 2710 and the lane line 2720 to an electrical power source 2780. In some embodiments, the power source may be a generator such as the generator illustrated in FIG. 16 of the present invention. The power source may be a high voltage power source to facilitate application of high voltage electrical pulses to the optional EPE for generating an electric field to the open pores of the diseased cells.

Handle 2790 at least partially houses electrical connector 2770 and is coupled to the proximal ends of the central probe and the access line to facilitate the penetration depth of the distal ends of the central probe and the access line. The handle 190 can provide a proximal termination point for various components (e.g., an access line, a first central lumen), a connection point for the central probe 2710 and the small aperture connector fitting 2795. The handle also serves as the primary user interface to the device and may contain one or more user input buttons electrically connected to the access line and/or optional electrodes for actuation or deployment thereof. The handle also houses an electrical connector 2770 that connects to a source of electrical power 2780. The handle allows control of the orientation and direction of the device, deployment and retraction of the access line, deployment and retraction of the central probe/needle, deployment and retraction of the optional electrodes, and remote triggering of the delivery of the electroporation pulse (optional). Additionally, as described above, the handle is configured to facilitate the penetration depth of the needle, the access line, and the electrode.

In some embodiments, handle 2790 is shaped for ease of use by a physician, e.g., with a molded handle portion or grip, optional illumination elements at the distal tip, a camera for viewing and recording the treatment site, biopsy forceps, tissue scissors, an engagement device, a suturing system, etc.

In addition, the electrodes 2750 and handle 2790 are preferably made of materials that can be sterilized and configured to similarly minimize microbial entrapment if the electrode array and wand housing are to be reused. In some embodiments, at least the electrode array is disposable, and in some embodiments, the entire handle is also disposable.

In some embodiments, as illustrated in fig. 25 and 26, the EP delivery device may further include a catheter shaft surrounding an outer surface of the central probe to support and protect the central probe during insertion into a body having tissue, as illustrated in fig. 8.

a. Electroporation electrode

As described above, EPE 2750 is electrically connected to EP power supply 2780. The electrical connector 2770 may include four or more conductive wires (depending on the number of EPEs) for communicating electrical signals from the power supply to each of the EPEs. These signals may include the pin voltage set point, pulse width, pulse shape, number of pulses, and switching sequence. As will be appreciated by those skilled in the art and described more fully below, the EP electrode may also function as a Capacitive Sensing (CS) or impedance sensing (EIS) electrode, in which case the second low voltage power supply is used with a suitable switching mechanism to allow for the delivery of a higher voltage EP signal, and then a lower voltage CS or EIS signal, as illustrated in fig. 1.

EPE electrode 2750 is formed from a conductive material, although an optional insulating coating may be used as discussed herein. The electrodes may be made of any conductive material capable of delivering the large instantaneous current density associated with the applied high voltage pulse, including but not limited to: certain metals and their oxides, including gold, platinum, palladium, silicon, aluminum; a metal oxide electrode comprising platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo) ₂ O ₆ ) Tungsten oxide (WO) ₃ ) And ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, and carbon paste). Preferably the electrode comprises AgCl, cobalt-chromium, titanium, stainless steel, platinum, gold or a metal with high electrical conductivity plated with gold or platinum.

In addition, the electrodes, the TM delivery device, and the rod housing are preferably made of materials that can be sterilized and configured to similarly minimize microbial entrapment in the event the electrode array and rod housing are to be reused. In some embodiments, at least and/or the TM delivery and electrode arrays are disposable, and in some embodiments, the entire rod housing is also disposable.

In some embodiments, such as when the distal end of the EPE is exposed for generating an electric field, but its proximal end may be coated with a non-conductive substance so as to limit the application of the electric field to only the distal end of the EPE adjacent to the tissue and not along the length of the electrode, e.g., allowing EP that is "deeper" in the tissue but not in "shallow" regions. In some embodiments, the EPE may have alternating regions of insulating material and bare electrode, as generally depicted in fig. 4A and 4B. In this embodiment, the electrodes may be coated with the same pattern, resulting in a more uniform electric field, or coated with different patterns, resulting in an asymmetric electric field. Similarly, the electrodes may have the same length or different lengths for all electrode configurations herein.

The pulsed electric fields generated by these partially insulated EPEs are concentrated during treatment primarily in the regions between and near the exposed tip portions at the distal ends of the electrodes, and are small in the regions between and near the insulated portions.

In some embodiments, the EPE is substantially of a length so as to completely surround the tissue to be treated. In a preferred embodiment, all sets of electrodes (the "array" of electrodes) have the same length within the array, but the use of different length electrodes may result in a modified and asymmetric electric field in some instances.

In many embodiments, the length of the electrodes ranges from 1mm to 20 mm. Note that this measurement is the insertion depth and not the total length of the electrode 2750; there will generally be a portion of the electrode extending up from the point of contact with the tissue and into the handle 2790 for attachment to the appropriate circuitry, holding the electrode in the correct spatial configuration, and so on.

In many embodiments, the width and cross-sectional shape of the electrode for insertion are configured to minimize pain. Thus, the width of the electrodes may be from about 0.5mm to 1mm to 20mm, with from 1mm to 15mm being preferred.

b. Therapeutic moiety delivery method

Various embodiments of the present invention are directed to methods for delivering a therapeutic moiety to cells in a region of target cells in a tissue using the delivery device 100 of the present invention.

In some embodiments, a method for delivering a therapeutic moiety to a region of target cells comprises providing a device for delivering a therapeutic moiety of any of the embodiments of the invention described herein to a region of target cells of a tissue. Fig. 33 is an illustration of a method for delivering a therapeutic moiety to a region of target cells of a tissue using an EP device according to the present invention.

In some embodiments, a method for delivering a therapeutic moiety to a cell may include inserting a central probe/needle (for example) 2710 into the cell. In some embodiments, such as illustrated in fig. 25 and 25, the EP device may be used with an endoscope to access a cavity of the body that is not readily accessible. The access wire 2720 is deployed from inside the central probe 2710 to outside the central probe 2710 via an exit port 2730 on the sidewall of the central probe 2710, thereby creating a fluid channel in the cell or tissue. The lane line 120 is then removed, the center probe 2710 is rotated and the lane line is again extended. Multiple probe deployments create fluid channels within the tumor. In the case of removal of the access line, the therapeutic agent is injected, thereby flowing into the fluid channel.

A method for delivering a therapeutic moiety to a region of a target cell according to an embodiment of the present invention further includes inserting the central probe 2710 into a diseased cell in the region of the target cell, actuating and extending an access line 2720 from the central lumen in an axial direction of the central probe 2710, and piercing the cell or tissue with a needle-shaped distal end of the access line. The method can further include forming an opening as a result of the piercing through which at least a portion of the access line 2720 enters the tissue and creates a fluid channel through which the therapeutic portion is delivered. The method can further include actuating a ramp 2760 integrally formed or coupled with the inner surface of the central probe 2710 and contacting the access line with the ramp in order to direct the trajectory of the access line through the exit port toward the distal end of the central probe 2710. Upon exiting the central probe, the access line 2710 extends to pierce the tissue and create an opening through which at least a portion of the access line enters the tissue to create a fluid channel for delivery of the treatment portion to the tissue. The access line can be retracted into the central lumen and then the treatment portion injected into the central probe by a syringe. Once injected into the central probe, the treatment portion travels out through the exit port and into the channel in the cell created by the insertion of the access line.

In other embodiments, the lane line may have a blade shape, and the method of creating a channel may further comprise rotating the lane line while in the cell to create a hollow cylindrical channel having a larger area for receiving a larger amount of the treatment portion.

VI, the adaptive control method of the invention

Various embodiments of the present invention are directed to adaptive control methods for controlling EP pulse parameters during EP of cells in tissue using an EP apparatus. Fig. 36 is a flow diagram illustrating a control routine for an adaptive control method for controlling EP pulse parameters during use of an EP system in accordance with the present invention, and fig. 37 is a flow diagram illustrating a step-ahead feedforward control routine for optimizing EP pulse parameters using the control routine of fig. 36 in accordance with the present invention. In some embodiments, the adaptive control method may be implemented using any EP device described herein. However, the methods of the present invention are not so limited, but may also be practiced on any of those EP systems and devices/applicators and any methods as outlined in, for example, U.S. provisional patent application Nos. 62/214,807, 62/214,872, 62/141,142, 62/141,182, 62/141,256, and 62/141,164, all of which are expressly incorporated herein by reference in their entirety, including, in particular, the figures, illustrations, and descriptions of the figures and components therein.

The devices, systems and methods of the present invention will improve the process of EP-based gene therapy. Current EP systems apply open-loop control systems that use static parameters that rely on a priori knowledge determined by preclinical studies in homogeneous syngeneic tumor models. However, preliminary data have shown that even in homogeneous tumors, the time required to apply an electrostatic field across the cell membrane follows a log-normal distribution. Even in homogeneous models, applying static parameters to different tumors results in a wide range of applied electrostatic fields across the cell membrane and in treatment variability. One potential remedy is to define static parameters for applying sufficiently long EP pulses covering 95% of the known film charging time. However, due to the variation in charging time, the average tumor will be over-treated by a factor of 4, increasing the likelihood of adverse effects such as necrosis and apoptosis. The present invention provides a solution to the aforementioned problems by implementing a closed-loop control system that uses feedback control based on tissue sensing to optimize the EP process with tumor-specific measurements taken before and between each EP pulse. In some embodiments, tissue sensing will be used to measure the membrane charge time for a particular tumor to adjust each EP pulse for optimal treatment. Constraint boundaries are applied to the EP pulse parameters to ensure feedback convergence. The requirements needed to implement a closed loop control system for enhanced EP are (1) the ability to apply an electrical force on the tissue to drive the tissue toward a desired state, and (2) the ability to measure the state of the tissue. This may be achieved by measuring the bioelectrical change due to the applied electrical excitation signal.

The feedback adaptive control method of the present invention employs a closed-loop feedback control mechanism to regulate EP by monitoring the physiological properties of the tumor before and between EP pulses. The physiological properties will be determined by fitting EIS tissue data to the equivalent circuit model described herein in real time with a non-linear least squares curve fitting routine. Fitting the data to a tissue model allows quantification of the integrity of the cell membrane, represented by CPE, for the tissue to be treated. The duration of the EP pulse is modulated based on CPE model fitting parameters, allowing EP to be stopped when the relative change in CPE parameters reaches a level associated with therapeutically beneficial pDNA expression. The control devices, systems, and methods of the present invention will allow a user to inject therapeutic molecules, characterize the baseline state of the tissue, deliver optimized EP pulses for the tissue, and stop the pulses when a relative decrease in film integrity is achieved. This removes any ambiguity associated with EP and ensures successful delivery of the immunotherapeutic gene regardless of changes in tumor properties. Thus, EIS represents a significant development in the hardware currently used for clinical intratumoral immunotherapy.

Various aspects of the present invention address the need for practical advances in EP by implementing a dynamic feedback control system as described above. In vivo EP for gene therapy has been used clinically for vaccination and oncology indications for many different tissue types and tumors. As described above, EIS is a low power technology capable of monitoring an organization in real time. This technique is performed by applying a series of low voltage excitation signals across a pair of electrodes and measuring the response current over a range of frequencies. The magnitude and phase of each applied excitation is then calculated and fitted to an equivalent circuit model of the tissue. The above illustrates a common equivalent circuit for an organization. In this model, a resistive element (R) _I And R _E ) Due to the intracellular and extracellular matrix, respectively, and the lipid structure is composed of Constant Phase Elements (CPE) _M ) And (4) showing. CPE (customer premises Equipment) _M Is a charge or capacitance (represented by Q) representing the lipid bilayer _M Represented) and a function of a scalar (represented by a) ranging from 0 to 1 representing the non-ideal properties of the capacitor. The time constant for charging the lipid bilayer was calculated as τ = (R) _I Q _m ) ^1/α Can be used to identify the optimal EP pulse duration prior to each treatment.

In some embodiments, an adaptive control method for controlling EP pulse parameters during Electroporation (EP) includes providing any of the EP systems described herein. Various embodiments of the EP system and apparatus of the present invention utilize the same electrodes to perform low power EIS measurements and high power EP pulses. The foregoing configuration is desirable because it reduces the number of electrodes required and directly measures tissue response. The adaptive control method further includes initializing EP pulse parameters for performing EP in the tissue, and the initialized EP pulse parameters are based at least in part on at least one trained model as illustrated in fig. 38. FIG. 38 is a graphical representation of an initial training phase of a model to estimate pulse parameters in accordance with the present invention. As previously described, the model may be a physics-based model, an empirical model, or a data-driven model. In some embodiments, the trained model is trained using empirical data observed during initial operation of the EP device using fixed EP pulse parameters. The model may be trained using a supervised learning routine using machine learning methods. In some embodiments, a particular implementation for the model prediction phase may be a decision support tree that generates a set of logical rules for parameter estimation and diagnosis according to the adaptive control method of the present invention.

In some embodiments, the present invention relates to a "single-step feedforward control". By "step-ahead feed-forward control" is meant that prior to application of the first EP pulse, the parameter estimation routine initializes initial control parameters for the first pulse based on a model trained in an initial training phase using empirical data from previously conducted experiments. These previously performed experiments may be based on, for example, tissue samples with tumors having similar characteristics as those of the current tissue to be subjected to the control method of the present invention. The initialization may be an initial training phase performed off-line. A parameter estimation routine (described more fully below) is first generated during an initial model training phase using, for example, empirical data collected from several experiments/trials. This can be done off-line by operating the system without any feed-forward or feedback control (fixed pulse parameters). Empirical data may include a variety of fixed pulse settings, resulting characteristics, and corresponding biological outcomes resulting from these experiments/trials. Based on previously trained models and measured features derived from tissue sensing measurements in an initial training phase and the identified tissue or tumor type in a diagnostic phase, the controller uses a parameter estimation routine to select the best parameters/conditions for the first EP pulse. These first pulse parameters are thus "fed forward" to be applied as first pulses for the control routine, in contrast to conventional EP systems and methods in which the parameters/conditions of the first pulses are based on fixed conditions. In this sense, the method of the present invention utilizes feed forward control to provide optimal EP parameters based on the sensed tissue type, in conjunction with feedback control to sense cellular conditions, such as the degree of penetration, and adjust the pulse parameters accordingly.

Fig. 41A and 41B are flowcharts illustrating a method for adaptive control of EP pulse parameters according to the present invention. As illustrated in fig. 41A, the adaptive control method further comprises applying voltage and current excitation signals to the cells from the signal generator 1530 using the ith electrode pair of the EP device 1540 and measuring the voltage and current across the cells and tissue corresponding to the applied excitation signals to obtain the dielectric and conductive properties of the cells and tissue, including but not limited to capacitance, resistance, and impedance. Here, i =1 for measurements taken across the first set of electrodes. In some embodiments, the current and voltage measurements may be made by a voltage sensor and a current sensor, such as illustrated in measurement device 1510 of the control system of fig. 15. Current and voltage sensors (which may be integrated into the electrodes of the EP device or separately included elsewhere in the control system of the present invention) act as transducers that sense the current and voltage across the cell membrane and detect any change in quantity and provide output signals to the controller 1505 for the controller to carry out the function corresponding to the signal received from the sensors, i.e., to predict the first pulse parameter. An excitation voltage signal is initially applied, and then applied between each set of EP pulses, e.g., between the first and second EP pulses, and a measurement is taken across the tissue. This signal may be a band limited signal. The corresponding current signal is measured. This sensor data is time-dependent and is saved internally for use during data pre-processing as described below.

The adaptive control method further includes obtaining sensor data from the measurement device 1510 corresponding to the results of the measured cell or tissue property, and processing the data into diagnostics and updated control parameters. In these embodiments, the pre-processing module 1550 of the controller 1505 pre-processes the data to separate desirable data from undesirable data. In some embodiments, the voltage and current sensors 1510 communicate signals to a controller 1505 of voltage and current measurements, and the controller derives impedance data from these measurements.

As illustrated in fig. 41A, a pre-processing module 1550 of controller 1505 pre-processes and separates the measured data into desirable data and undesirable data. Controller 1505 may run algorithms to process data obtained from various measurements and stored internally, which may allow plotted curves and various other statistical analyses to be completed in order to find a set of EP parameters that yields the best EP results. In some embodiments, undesirable data is stored in the memory module in order to mark subsequently collected data of similar nature as "undesirable" data as an additional safeguard.

In some embodiments, the data pre-processing may include data mining. Often the data gathering method is loosely controlled, resulting in out-of-range values, impossible data combinations, missing values, etc., so analyzing data that has not been carefully screened for such undesirable data can produce misleading results. Thus, the data pre-processing carried out by the controller of the present invention provides the necessary safeguards for the quality of the representation of the data. To this end, the sensor data is cleaned by removing outliers, out-of-range values, missing values, removing bias, scaling, cross-correlation, and applying a de-noising routine. In some embodiments, a sensor validation routine is used to determine or evaluate the data quality prior to the controller extracting features to estimate improved and more ideally optimized EP pulse parameters.

In some embodiments, the pre-processing module of the controller may pre-process the data using any of:

denoising filter-a digital filter that removes noise from the sensor signal. The filter may be implemented as an Infinite Impulse Response (IIR) or Finite Impulse Response (FIR) filter. This may also be implemented as an analog filter or as part of the EP circuit. Unbiasing-the AC signal measured by the sensor can be pre-processed by removing the DC bias from each of the signals. Scaling-the data may be based on a normalized value such as the standard deviation. Median filtering — the data can be filtered using nonlinear digital filtering techniques. Outliers- -data can be processed to remove extreme values by identifying one or more values from the rest of the data set that are outside of a specified number of standard deviations. Sensor validation-the controller may implement a routine by running an algorithm that analyzes the quality of the measured data using statistical measures such as standard deviation, number of outliers, skewness and kurtosis, or any other known statistical measure for analyzing the quality of the data. For example, if the standard deviation of the data exceeds a threshold, the data is marked as "undesirable data" or an unusable data set.

The adaptive control method of the present invention further includes extracting relevant features from the desired data by a feature extraction module 1570. "feature" means a value derived from preprocessed data that is intended to be informative and non-redundant based on various characteristics of the preprocessed data. The characteristics of the system may be estimated and controlled, thus proving that the system can be stabilized by controller 1505.

In some embodiments, the feature extraction module is configured to execute certain software instructions for deriving relevant features from the preprocessed desired data using a computation routine. The characteristics of the pre-processed consensus data, including but not limited to data descriptive statistics, data descriptive models, time-independent transforms, time-series transforms, domain-dependent feature extraction, may be obtained using a computation routine.

In some embodiments, data descriptive statistics for sensor data may include, but are not limited to, mean, standard deviation, peak-to-peak, root Mean Square (RMS), variance, kurtosis, crest factor, correlation coefficient, autocorrelation, and cross-correlation. For events, data descriptive statistics may include counts, incidence, duration, and time delays. The data descriptive model may comprise a distribution model, such as a parametric distribution, a histogram, a regression model (using model parameters or modeling errors): curve fitting, autoregressive (AR) models, classification/clustering models (using class labels as features), sequence matching possibilities, pattern recognition classifiers (fisher's discriminant, bayesian theorem), time independent transforms may include explicit mathematical operations such as difference, sum, ratio, logarithm, power n, principal component analysis, and independent component analysis. The time series transform may include a frequency domain, a time-frequency domain, and a wavelet domain. Domain-dependent feature extraction may include physics-based features such as expected input-output or output-output relationships, derived hidden states, and special procedures for data processing such as operating mechanism segmentation, and envelope analysis.

In some embodiments, the features are derived from a parametric model fit of the magnitude ratio or phase difference of the excitation voltage and current signals. These data include, but are not limited to, intracellular resistance, extracellular resistance, solution resistance, film capacitance, admittance, constant phase element index, and charge time constant. Feature extraction by the feature extraction module may comprise determining the capacitance or impedance of the cell membrane of the cell resulting from the applied excitation signal. In these embodiments, the impedance may be determined from measurements of the dielectric and conductive properties of cells and tissues resulting from the applied excitation signal by applying a band limited signal that is repeated over a fixed frequency range. The dielectric and conductive properties of the cells or tissues are determined by the magnitude ratio and phase difference of the excitation voltage and current applied to the cells or tissues. Controller 1505 may calculate the magnitude and phase of each applied stimulus and fit these to the equivalent circuit model of the tissue described above. In the model, the resistive element (R) _I And R _E ) Due to the intracellular and extracellular matrix, respectively, and the lipid structure is composed of Constant Phase Elements (CPE) _M ) And (4) showing. CPE (customer premises Equipment) _M Is a charge or capacitance (represented by Q) representing the lipid bilayer _M Represented) and a function of a scalar (represented by a) ranging from 0 to 1 representing the non-ideal properties of the capacitor. The time constant for charging the lipid bilayer was calculated as τ = (R) _I Q _m ) ^1/α It can be used by the pulse parameter estimation module to estimate the optimal EP pulse duration before each treatment.

The magnitude ratio and phase difference between the excitation voltage and current signals applied to cells and tissue is determined by cross-correlating the excitation voltage and current signals with known reference signals stored in the memory module. Examples of features include (but are not limited to) the following: a) A magnitude ratio and a phase difference value of the excitation voltage and current signals at a fixed frequency; b) At least one of an average, median, maximum, and minimum of: i) A magnitude ratio or phase difference of the excitation voltage and current signal magnitudes over a narrow frequency band, ii) a magnitude ratio or phase difference of the excitation voltage and current signal magnitude phases over a wide frequency band; c) Curvature, slope and noise of the magnitude ratio or phase difference versus frequency of the excitation voltage and current signals; d) A constant phase base parameter; e) High frequency resistance (e.g., at 100 Hz), low frequency resistance (e.g., at 1 kHz); and f) a capacitance.

In some embodiments, the control method of the present invention includes applying, by the diagnostic module, at least a portion of the relevant features of the desired data to at least one trained diagnostic model, as illustrated in fig. 40. A priori tissue diagnostics is important in the prediction of successful EP. As illustrated in fig. 40, the features were used as inputs to a series of diagnostic models for (i) tissue detection, (ii) tumor type detection, (iii) injection detection, and (iv) penetration detection. Based on the results of these models, the system will (i) terminate the treatment due to infiltration, (ii) proceed to estimate the next pulse parameters, or (iii) stop and alert the operator to a diagnostic event (e.g., no tissue detected, no tumor detected, no infusion detected). One or more statistical inference routines (e.g., bayesian reasoners) will be used to combine or fuse the multiple features for each diagnostic module. The system will include several diagnostic modules to make decisions on the control inputs (applying pulses). In some embodiments, the control method comprises fitting or applying, by the controller, the derived features to a trained diagnostic model, and observing a fit of the data, wherein a poor fit or correlation is an indicator of a diagnostic problem, such as improper electrode placement, e.g., in necrotic or fibrotic tissue, erosion of the electrode. In some embodiments, the criterion for tissue fitting to a model, e.g., a CPE-based tissue model, is R ² >0.98. The diagnostics module 1580 of controller 1505 generates a diagnostic response based at least in part on the results of the fitting or applying as described above, wherein the diagnostic response includes tissue detection, tumor type detection, needle placement detection, cell penetration detection, co-location diagnosis, pulse verification, and repetitive pulse diagnosis.

The diagnostics routine described in detail above plays an important role in the control method of the present invention. One area where this is particularly important is co-location detection. Overlapping the electric field and the implant is critical to the success of the EP process. The electrical measurements ensure that abnormal conditions do not interfere with the treatment. Examples of problems causing poor co-localization include, but are not limited to, deeper than effective electric field injection, deflection of the injection elements, and biological anomalies in the tissue or cells. Experiments and studies performed have shown that good co-localization is characterized by at least a 10% decrease in solution resistance. The present invention is directed to achieving good or desirable co-location, at least in part, by integrating a treatment portion delivery device with an EP electrode in a single EP device or applicator. The diagnostic routine of the present invention performed by the diagnostics module ensures that EP is performed only after good co-localization is observed, tissue is detected, tumor is detected, and an injection is detected. When the aforementioned conditions are met, the controller then applies the correlation features to the CPE-based tissue model to estimate the initial pulse parameters.

In some embodiments, as described above, the adaptive control method further comprises estimating, by the pulse parameter estimation module, the first pulse parameter based on a result of applying the relevant features to the CPE-based tissue model after execution of the diagnostic routine and feature extraction described above. That is, if tissue is detected, if a tumor is detected, and if an injection is detected, initializing EP pulse parameters is based on the at least one trained model and the measured features to estimate an improved or ideally optimized first EP pulse parameter. In some embodiments, as illustrated in fig. 36 and 37, features combined with past features will be used to determine future pulse parameters. The estimator may include a state space estimation, artificial neural network, autoregressive (AR), and autoregressive moving average (ARMA) estimator.

In some embodiments, the control method further comprises applying the first EP pulse based on estimating the improved/optimized first pulse parameter. Various embodiments of pulse sequences are illustrated in fig. 41A and 41B, where i = pulse sequence (i =0 for the excitation signal, i =1 for the first applied EP pulse), and N = number of electrode pairs. The adaptive control method may further comprise predicting subsequent EP pulse parameters after the first EP pulse has been applied using a trained CPE-based model based on previous EP pulse parameters and changes in at least one of voltage and current measurements of the cell's response to the first EP pulse and said characteristics between applied EP pulses. As illustrated in fig. 41B, the above-described tissue sensing routine repeats between applied EP pulses until optimized EP pulse parameters are achieved or until a pulse limit is reached.

In some embodiments, the control method may further comprise a) applying a subsequent EP pulse based on the predicted subsequent EP pulse parameters, and b) repeating the application of the voltage and current excitation signals to the cells and tissue, repeating the measuring of the cells or tissue, repeating the obtaining of data and separating desirable data from undesirable data; the extraction of relevant features is repeated and the applying is repeated until either i) a predetermined limit on the number of EP pulse sequences or cycles of EP pulses is reached, or ii) the diagnostic response prompts a diagnostic decision to terminate the adaptive control method, as illustrated in fig. 41A and 41B. In some embodiments, the control method may terminate and no further EP pulses are applied when the time constant drops by 50%. At this point, expression in all groups was statistically determined to be significantly different from the control. As described above, the duration of the EP pulse is modulated in accordance with CPE-based model fitting parameters, thus stopping EP when the relative change in CPE parameters reaches a level associated with therapeutically beneficial pDNA expression. This technique would allow the clinician to inject therapeutic molecules, characterize the baseline state of the tissue, deliver an optimized EP pulse for that tissue, and stop the pulse when a relative decrease in membrane integrity is achieved. This removes any ambiguity associated with EP and ensures successful delivery of the immunotherapeutic gene regardless of changes in tumor properties.

Therapeutic moieties for delivery

The present invention provides devices and methods for improved delivery of a therapeutic moiety to cells in a tissue of a patient. Generally, the system of the present invention is used to treat diseased or abnormal tissue, such as cancerous tissue. The term "cancer" encompasses a large number of diseases characterized generally by inappropriate, abnormal, or excessive cell proliferation. The device is intended for use in patients suffering from cancer or other non-cancerous (benign) growths. These growths themselves may manifest as any of the following: a lesion, a polyp, a neoplasm (e.g., a papillary urothelial neoplasm), a papilloma, a malignant disease, a tumor (e.g., a clade tumor, a tumor of the hepatic portal region, a non-invasive papillary urothelial tumor, a germ cell tumor, an ewing tumor, an astrjin tumor, a primitive neuroectodermal tumor, a leydig cell tumor, a wilms tumor, a seltory cell tumor), a sarcoma, a carcinoma (e.g., a squamous cell carcinoma, a cloacal carcinoma, an adenocarcinoma, an adenosquamous carcinoma, a cholangiocarcinoma, a hepatocellular carcinoma, a traumatic papillary urothelial carcinoma, a squamous urothelial carcinoma), a mass, or any other type of cancer or non-cancerous growth. Tumors treated with the devices and methods of embodiments of the present invention may be any of non-invasive, traumatic, superficial, papillary, flat, metastatic, localized, single-centered, multi-centered, low-grade, and high-grade. Examples of cancers include, but are not limited to, breast, colon, prostate, pancreas, skin (including melanoma, basal cell and squamous cell), lung, ovary, kidney, brain, or sarcoma, adrenal cortex, anus, bile duct (e.g., peritoneal, distal bile duct, intrahepatic bile duct), bladder, benign and cancerous bone (e.g., osteoma, osteogenic osteoma, osteoblasta, osteochondroma, hemangioma, chondroamphoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancers (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglioglioma, schwannoma, blastoma, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, small She Yuanwei carcinoma, gynecomastia), castleman's disease (e.g., giant lymph node hyperplasia, follicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenocarina, papillary serous adenocarcinoma, clear cells), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid (e.g., choriocarcinoma, meningioma destruction), hodgkin's disease, non-hodgkin's lymphoma, kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma), laryngeal and hypopharyngeal cancers, choriocarcinoma (e.g., hemangioma, hepatoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal and paranasal sinus cancers (e.g., nasal glioma, midline granuloma), nasopharyngeal carcinoma, neuroblastoma, oral and oropharyngeal cancers, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma multiforme), salivary gland cancer, skin cancer (melanoma and non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminoma germ cell carcinoma), thymus cancer, thyroid cancer (e.g., follicular carcinoma, degenerative carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulval cancer, and uterine cancer (e.g., uterine leiomyosarcoma). Thus, cancerous tissue, including skin tissue, connective tissue, adipose tissue, and the like, can be treated using the system of the present invention. These cancers may be caused by chromosomal abnormalities, degenerative growth and development disorders, agents that promote cell division, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of genes, alterations in gene expression, or carcinogenic agents.

The term "treatment" includes, but is not limited to, inhibition or reduction of cancer cell proliferation, destruction of cancer cells, prevention of cancer cell proliferation or prevention of malignant cell initiation, or suppression or reversal of progression of transformed premalignant cells to a malignant disease, or amelioration of the disease. The term "subject" or "patient" refers to any animal, preferably a mammal such as a human. Livestock use is also intended to be encompassed by the present invention.

The systems and methods of the present invention deliver a therapeutic moiety to cells in tissue in an electroporation zone. By "therapeutic moiety" or TM herein is meant a moiety suitable for electroporation that is capable of treating diseased tissue that contains a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, interleukin, or other therapeutically active agent. The therapeutic moiety may be a small molecule drug, a nucleic acid (including those encoding a therapeutic target protein), or a biologically active protein (including polypeptides and peptides), as more fully outlined herein.

In some embodiments, TM is a drug; the drugs contemplated for use in the methods of the invention are generally chemotherapeutic agents having anti-tumor or cytotoxic effects. These drugs or agents include bleomycin, neocarzinostatin, suramin, doxorubicin, carboplatin, paclitaxel, mitomycin C and cisplatin. Other chemotherapeutic agents will be known to those skilled in the art (see, e.g., the Merck index). Electroporation facilitates the entry of bleomycin or other similar drugs into tumor cells by creating pores in the cell membrane. This local delivery provides significant benefits because the normal systemic toxicity typically associated with these drugs is minimized via local administration by the EP methods herein.

In some embodiments, the TM is a biomolecule, including nucleic acids and proteins.

In some embodiments, the TM is a nucleic acid. Generally, a TM that is a nucleic acid has two distinct functional types. In one embodiment, the nucleic acid encodes a protein for treating a disease; in other embodiments, the nucleic acid is a TM, e.g., when the nucleic acid is an siRNA or snRNA. By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein is meant at least two nucleosides covalently linked together. <xnotran> , , , , (Beaucage , tetrahedron 49 (10): 1925 (1993) ; letsinger, 《 (J.Org.Chem.) 》 35:3800 (1970); sprinzl , 《 (Eur.J.Biochem.) 》 81:579 (1977); letsinger , 《 (Nucl.Acids Res.) 》 14:3487 (1986); sawai , 《 (Chem.Lett.) 》 805 (1984), letsinger , 《 (J.Am.Chem.Soc.) 》 110:4470 (1988); Pauwels , 《 (Chemica Scripta) 》 26:141 91986), (Mag , 《》 19:1437 (1991); 8978 zxft 8978 ), (Briu , 《》 111:2321 (1989), O- ( Eckstein, 《 : (Oligonucleotides and Analogues: A Practical Approach) 》, ), ( Egholm, 《》 114:1895 (1992); meier , 《 (Chem.Int.Ed.Engl.) 》 31:1008 (1992); nielsen, 《 (Nature) 》 365:566 (1993); carlsson , 《》 380:207 (1996), ). (Denpcy , </xnotran> Journal of the national academy of sciences (proc.Natl.Acad.Sci.USA) 92; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; kiedrowshi et al, applied to International publication on the chemistry (Angew. Chem. Int. Ed. English) 30 (1991); letsinger et al, J.Chem.Soc. 110 (1988); letsinger et al, nucleotide and Nucleotide (Nucleoside & Nucleotide) 13 (1994); ASC conference 580, carbohydrate modification in Antisense studies (Carbohydrate modification in Antisense Research) 2 and 3, Y.S.G. huui and P.Dan Coje et al, modification of Carbohydrate in Antisens Research, sanchikun & R. J.R. Pat. No. 5 & gt 5 & J.23, and Biochemfs.11 (Biochex. J.11, J.11 & N.R. Pat. J.; carbohydrate modification, J.11 & N.J. Pat. J. 11 & A. Biochex.A.A. 23 (Biochem., USA No.. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al, reviews of chemical sciences (chem. Soc. Rev.) (1995) pp 169-176). Several nucleic acid analogs are described by Rawls in chemical and engineering News (C & E News) 1997, 6, 2, page 35. All of these references are hereby expressly incorporated by reference herein. These modifications of the ribose phosphate backbone can be done to increase the stability and half-life of these molecules in physiological environments, for example when the nucleic acid is siRNA or the like.

In many embodiments, the nucleic acids of the invention are contained within one or more expression vectors containing additional nucleic acid sequences that confer functionality to the expression vector, including but not limited to promoters, regulatory sequences, and the like.

In some embodiments, the nucleic acid is DNA or RNA encoding a therapeutic protein moiety, such as an antibody and an interleukin.

In some embodiments, the nucleic acid encodes an immunostimulatory cytokine, as summarized herein. The phrase "immunostimulatory cytokine" includes an interleukin that mediates or enhances an immune response to a foreign antigen comprising a viral, bacterial, or tumor antigen. The innate immune stimulating interleukins can comprise, for example, TNF- α, IL-1, IL-10, IL-12, IL-15, type I interferons (IFN- α and IFN- β), IFN- γ, and chemokines. Adaptive immunostimulatory interleukins include, for example, IL-2, IL-4, IL-5, TGF- β, IL-10 and IFN- γ. Examples of immunostimulatory interleukins are provided in table 1 below.

Table 1: immunostimulatory interleukin accession number

An immunostimulatory cytokine particularly suitable for use in the present invention is IL-12.

In some embodiments, the nucleic acid encodes a therapeutic antibody. In general in this embodiment, there are two nucleic acids electroporated into the tissue, one encoding the heavy chain and one encoding the light chain. In some cases, these may be in a single expression vector or two expression vectors may be used, as described more fully below.

The term "antibody" is used generally. Antibodies suitable for use in the present invention may take a variety of forms as described herein, including conventional antibodies as well as antibody derivatives, fragments and mimetics as described below. Conventional antibody building blocks typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention relates to the IgG class, which has several subclasses, including but not limited to IgG1, igG2, igG3 and IgG4, wherein the former are particularly suitable for use in many applications, especially oncology. Thus, "isotype" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It is understood that therapeutic antibodies may also comprise hybrids of the same type and/or subclass.

In some embodiments, the antibody is a full length antibody. By "full length antibody" herein is meant a structure that constitutes the native biological form of the antibody, comprising variable and constant regions, optionally comprising one or more amino acid modifications as known in the art. Alternatively, the antibody may be of a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively. Specific antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable (Ward et al, 1989 nature 341, incorporated by full reference), (v) isolated CDR regions, (vi) F (ab') 2 fragments, including two bivalent fragments linking Fab fragments, (vii) single chain Fv molecules (scFv), in which the VH and VL domains are linked by a peptide linker that allows association of the two domains to form an antigen binding site (Bird et al, 1988 nature 242, 423-426, huston et al, 1988 journal of the american college of sciences 85, 5879-5883, incorporated by full reference), (viii) bispecific single chain Fv (WO 03/11161, hereby incorporated by reference), and (ix) "bifunctional antibodies" or "trifunctional antibodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al, 2000 Methods of enzymology (Methods 326, enzymology) enzl 461-479, incorporated by full reference, kogaku 6448, 1988). Antibody fragments may be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking VH and VL domains (Reiter et al, 1996 "Nature biotechnology (Nature biotech.)" 14, incorporated by full reference).

As will be appreciated by those skilled in the art, there are a wide variety of suitable therapeutic antibodies that can be used in the present invention, depending on the type and location of the cancer. Suitable therapeutic antibodies include, but are not limited to, human, humanized or chimeric antibodies for therapeutic use in humans, including currently approved antibodies that are the same as or similar to molo, abciximab, rituximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab, alemtuzumab, iso Bei Mo mab, adalimumab, omalizumab, tositumomab, efletuzumab, cetuximab, bevacizumab, natalizumab, ni Wo Shankang, parislizumab, and galituximab MPDL328OA (ROCHE), as well as those antibodies in clinical development, particularly oncology applications.

In addition, the present invention provides EP methods and devices for delivering therapeutic antibodies to immune checkpoint inhibitors. As used herein, an "immune checkpoint" molecule refers to a group of immune cell surface receptors/ligands that trigger T cell dysfunction or apoptosis. These immunosuppressive targets attenuate excessive immune responses and ensure self-tolerance. Tumor cells utilize the inhibitory effects of these checkpoint molecules. The immune checkpoint target molecules include, but are not limited to, the checkpoint targets described in table 2.

Table 2: checkpoint target accession number

The phrase "immune checkpoint inhibitor" encompasses molecules that prevent immune suppression by blocking the effects of immune checkpoint molecules. Checkpoint inhibitors may include antibodies and antibody fragments, nanobodies, diabodies, scfvs, soluble binding partners for checkpoint molecules, small molecule therapies, peptide antagonists, and the like. The inhibitors include, but are not limited to, the checkpoint inhibitors described in table 2.

In some embodiments, the EP methods and devices are used in combination therapy, e.g., for delivery of two different TM's of higher efficacy. As will be appreciated by one of skill in the art, the combination can be any of the TMs outlined herein, including but not limited to a) nucleic acids encoding therapeutic biomolecules (including expression vectors as described more fully below) and small molecule drugs, such as the plastids encoding IL-12 and drugs outlined above; b) A first nucleic acid encoding a first therapeutic biomolecule and a second nucleic acid encoding a second therapeutic biomolecule (e.g., two nucleic acids encoding an expression vector for IL-12 and encoding an anti-immune checkpoint inhibitor antibody as described herein), and c) a first nucleic acid encoding a first biomolecule and a second protein molecule, e.g., an anti-immune checkpoint inhibitor antibody; and d) two small molecule oncology drugs.

In some embodiments, the EP methods and devices of the present invention are used in a combination immuno-oncology therapy. In this embodiment, a combination therapy of an immunostimulatory cytokine therapy (as above) and a checkpoint inhibitor is administered to the patient.

In one embodiment, the immunostimulatory cytokine is administered in the form of a plastid containing a nucleic acid encoding the immunostimulatory cytokine, and the checkpoint inhibitor is administered as a protein (e.g., an antibody to the checkpoint inhibitor) into cells and tissues.

In another embodiment, the immunostimulatory cytokine is administered in the form of an expression vector plasmid containing a nucleic acid encoding the immunostimulatory cytokine and the checkpoint inhibitor is administered similarly to one or more expression vectors comprising a first nucleic acid encoding a heavy chain of an anti-checkpoint inhibitor antibody and a second nucleic acid encoding a light chain of an anti-checkpoint inhibitor antibody.

In this embodiment, one, two or three carriers may be used: if one is used, it contains coding sequences (and appropriate regulatory sequences) to express the immunostimulatory cytokine and the heavy and light chains of the anti-checkpoint inhibitor antibody. Alternatively, three expression vectors may be used, each encoding one of the above. Two expression vectors, one containing a single component (e.g., an immunostimulatory cytokine) and the other containing two components (e.g., the heavy and light chains of an anti-immune checkpoint inhibitor antibody) may also be used.

In addition, small molecule drugs may also be delivered in any combination of the above.

Furthermore, administration of anti-checkpoint inhibitors (and/or small molecule drugs as outlined above) may be done systemically rather than as EP therapy to achieve efficacy as well.

Administration of combination therapy can be achieved by electroporation alone or in combination with systemic delivery.

Other contemplated combination therapies are checkpoint inhibitors in combination with: TLR promoters (e.g., flagellin, cpG); IL-10 antagonists (e.g., anti-IL-10 or anti-IL-10R antibodies); TGF- β antagonist, CD3 promoter; telomerase antagonists, and the like.

VIII example

Example 1:

OncoSec constructs an EP generator a illustrated in fig. 16, which is capable of performing real-time feedback control based on EIS data before and between each EP pulse. This system is capable of outputting a minimum of 10V and a maximum of 300V with pulse durations ranging from 100 mus to 10 ms. The EIS data captured before and between pulses were obtained for 10 data points acquired every ten-fold range over the range of 100Hz to 10 kHz. The acquisition of EIS data over this spectrum is achieved in 250ms, which is fast enough to: (1) Executing a routine to determine a time constant for a next pulse; (2) storing the EIS data for post-analysis; and (3) does not interrupt clinically used EP conditions. The data gathered from the EIS system is fitted to the above tissue impedance model in real time using an embedded Advanced RISC Machine (ARM) microprocessor (STM 32F407, ST microelectronics). When operating in a feedback mode, characteristics of this data may be used to control parameters associated with the EP process. The custom generator interfaces with a variety of standard EP applicators, and will support up to 6 electrodes. The solid state repeater is used to switch between a high voltage EP pulse circuit and a low voltage EIS interrogation circuit. To allow hands-free operation of the generator, a foot pedal is added to trigger, pause or abort the EP process. An image of this generator and its accessories is shown in fig. 16.

Unpublished studies performed in vivo investigated the effect of varying pulse widths based on time constant data acquired from EIS spectra. These studies were performed on MC38 tumors implanted in the flank skin of 8-week old albino B6 mice. Tumor volume averaged 75mm at treatment ³ . Tumors were injected with 50 μ g of pDNA encoding luciferase protein under the control of CMV promoters. The technical prototype applicator C shown in fig. 16 containing the EP device was used to perform both injection and EP. This applicator contains two EP electrodes around a central infusion lumen; the infusion lumen is retracted during EP. Electroporation was performed at an electric field strength of 350V/cm, and the pulse width was modulated in real time from 0.1 to 20.0 times the time constant calculated from EIS data collected before pulsing each MC38 tumor. A total of 8 pulses were applied to each tumor as this had been previously associated with a high degree of transfection. 15mg of 200. Mu.l prepared in DPBS by infusionA/ml D-fluorescein solution and in vivo optical imaging was performed to obtain luminescence data at 48 hours. Summary data from this experiment are shown in fig. 42A to 42D.

Figure 42A illustrates the distribution of% applied electric field across the lipid bilayer versus time constant. Fig. 42B illustrates the distribution of time constants measured prior to EP. Fig. 42C illustrates the effect of modulating the pulse width based on pre-pulse EIS data, where the pulse duration was set at a multiple of the time constant for each tumor. Fig. 42D illustrates data showing the relative change in the calculated time constant after EP with respect to the resulting luminescence. Data found to be statistically significant at α =0.05 are indicated by asterisks.

These data show that pDNA expression depends on the applied pulse width. The original assumption was that as the applied pulse width increased relative to the measured time constant, the percentage of the electric field applied across the lipid bilayer would increase according to figure 42A. This is particularly important because the time constants calculated from the EIS data prior to each EP treatment follow the log normal distribution shown in fig. 42B. The results of the experiment in fig. 42C show that as the applied pulse width increases relative to the measured time constant, the resulting luminescence also increases. This phenomenon supports the hypothesis that the measured expression reaches an upper limit because the capacitor approaches charge saturation at a 5-fold time constant. Data sets acquired above both time constants experienced significantly (p < 0.05) higher luminescence when compared to injection alone. As the pulse width becomes longer and more energy is dissipated through the tissue, the expression will begin to weaken as irreversible tissue damage occurs.

In addition, this experiment shows a potential criterion to stop the EP process before reaching the previously determined number of terminal pulses. As the cell membrane begins to penetrate, its ability to hold charge decreases, which in turn causes a decrease in the time constant associated with charging the CPE. This theory is supported by the high correlation observed between the change in time constant and the measured luminescence. Tumors with a time constant reduction of more than 20% were associated with significantly (p < 0.05) higher expression of pDNA. This measurement can be used to stop the pulsing process when conditions for successful gene therapy exist. Interestingly, the group with a short pulse duration causes an increase in the time constant due to the compression of the lipid bilayer causing the capacitance to increase. For this study, we propose to utilize a technical prototype generator to explore variables for controlling EP and demonstrate this technique in homogeneous and heterogeneous tumors. We hypothesized that EP-based gene delivery can be optimized for each tumor by measuring tissue properties and adjusting each applied pulse width. Interrogation of real-time changes in the capacitance of the membrane will result in (1) reproducible transfection efficiencies; (2) increased duration of gene expression; (3) enhanced therapeutic efficacy; and (4) reduced tissue damage.

Example 2:

experiments were performed to determine whether Electrochemical Impedance Spectroscopy (EIS) could distinguish between data acquired in fibrotic or necrotic tissue and data acquired in healthy tissue. In these experiments, the liver of a 9-month old transgenic mouse expressing human platelet-derived growth factor C (PDGF-C) was used to represent fibrotic tissue. At this age PDGF-C transgenic mice are known to have significantly fibrotic, fatty infiltrates and enlarged livers with dysplastic cells. Data obtained from the livers of transgenic animals were compared to healthy or wild-type 3 month old C57BL/6J livers. To obtain this data, mice were anesthetized to allow surgical access to the liver. Parallel electrodes spaced 3mm apart were inserted 5mm into the left lateral lobe of the liver. The data collected from this experiment were fitted to a constant phase element model to electrically represent biological tissue. Parameters derived from model fitting reveal an increase in hepatic fibrosis leading admittance, an increase in the calculated time constant, and a decrease in the constant phase element. These data are shown in fig. 43.

Example 3:

a second experiment was performed to determine if EIS could detect the presence of the injection in tumor tissue. In this experiment, tumors were implanted in subcutaneous tissue by injecting 106MC38 cells with 50 μ Ι of phosphate buffered saline on the flank skin of 8-week-old albino B6 mice. After approximately 10 days, the tumors reached an average volume of 100mm 3. At this point a two-electrode applicator with a central infusion lumen was inserted 7mm into the tumor. Baseline EIS measurements of the initial condition of the tumor were then made. After this measurement, a 50. Mu.l volume of a 1mg/ml solution of plastid DNA prepared in physiological saline was injected into the tumor. A second EIS measurement is performed after the injection. These data are again fitted to a constant phase element model to represent biological tissue. At least a 10% drop in solution resistance was observed after injection into the tumor with the plastid DNA solution. Figure 44 provides a histogram summary of the percent reduction in solution resistance observed from the model fitting parameters after injection of plastid DNA.

Example 4:

in addition to detecting viability of tissue and presence of an implant, EIS may also inform the user of the optimal pulse width for performing electroporation. To demonstrate this, a study was performed that varied the pulse width based on time constant data acquired from model fitting of EIS spectra. This study was performed with MC38 tumors implanted in the flank skin of 8-week old albino B6 mice. Tumor volume averaged 75mm at treatment ³ . Tumors were injected with 50 μ g of pDNA encoding luciferase protein under the control of CMV promoters. A two-electrode applicator with a central infusion lumen is used to perform both the infusion and EP. The infusion lumen is retracted from the tumor during EP. Electroporation was performed at a field strength of 500V/cm and the pulse width was modulated around the mean time constant obtained a priori from 10 tumors. The time constant for this averaging calculation was 0.50ms, and the pulse widths selected for this experiment were 0.1, 0.5, 2.0, and 10.0 times the average time constant. A total of 8 pulses were applied to each tumor. Luminescence data were obtained at 48 hours after injection of 200. Mu.l of a 15mg/ml solution of D-fluorescein prepared in D-PBS. This data was collected by in vivo optical imaging. The data from this experiment show the maximum rise in luminescence for tumors treated with a 10-fold mean time constant or a total pulse width of 5 ms. In addition, these data show that the groups treated with a two or more time constant had a significant increase in luminescence compared to the injections alone. Summary data from this experiment is shown in fig. 45.

Example 5:

following the experiment conducted in example 4, a study was performed to determine whether EIS could be used in real time to enhance the optimal pulse width for each individual tumor. This would allow each electroporation sequence to be adjusted according to the initial conditions of each individual tumor. The MC38 tumor was again implanted in the flank skin of 8-week old albino B6 mice. When the tumor reached 75mm3, the tumor was injected with 50 μ g of pDNA encoding luciferase protein. The same two-electrode applicator with a central infusion lumen was used to perform both infusion and EP. For this experiment, the field strength was reduced to 350V/cm and the pulse width was modulated in real time using time constants calculated for each tumor being treated. The pulse width is modulated from 0.1 to 20.0 times the calculated time constant. A total of 8 pulses were applied to each tumor. Luminescence data were obtained by in vivo optical imaging by injecting 200. Mu.l of a 15mg/ml D-fluorescein solution over 48 hours. The data from this experiment showed a significant increase in luminescence for all tumors treated with a 2.0-fold and above time constant. No statistical differences were observed between groups at 5.0, 10.0 and 20.0 times the calculated time constant. Data from this experiment is shown in figure 46.

Post-processing of the data acquired during the course of this experiment indicates a potential criterion to stop the EP process before reaching the previously determined number of terminal pulses. As the cell membrane begins to penetrate, its ability to hold a charge decreases, which in turn causes a decrease in the time constant associated with charging CPE. This theory is supported by the high correlation observed between the change in time constant and the measured luminescence. Tumors with a time constant reduction greater than 20% were associated with significantly higher expression of pDNA. This measurement can be used to stop the pulsing process when conditions for successful gene therapy exist. Interestingly, the groups with short pulse duration cause an increase in the time constant due to the compression of the lipid bilayer which causes an increase in capacitance. These data are shown in fig. 47.

Example 6: (expedition)

Goal (goal 1) is to evaluate feedback parameters that lead to the desired outcome of intratumoral immunotherapy. Based on preliminary studies, EP integrated with EIS feedback control has the potential to reduce the rate of change between treatments. To assess pDNA expression and control the histological effects of EP based on changes in the calculated time constants, in vivo tumor studies will be performed in a homogeneous contralateral murine melanoma model. Briefly, B16/OVA cells (1 x 106/site) will be implanted subcutaneously in the flank of B6 mice (n = 10/group). When tumors reached a volume of 75mm3, they were injected with dual reporter plasmids (1 mg/ml, 50 μ Ι per tumor) that expressed both luciferase and mCherry. This would allow non-invasive, longitudinal bioluminescence imaging as well as spatial cell-specific gene expression. The tumor will be pulsed with FCEP using a two-electrode applicator C in fig. 16. The electrodes will be operated at 350V/cm with pulse widths set at five calculated time constants for each individual pulse. The cell will continue to receive EP pulses until a relative decrease of 20%, 40%, 60%, or 80% of the time constant is reached. The operating limits of the generator will be set to ensure safety, where the maximum pulse width allowed will be fixed at 10ms and the maximum pulse will be set at 10. Control animals for this experiment will contain no treatment, pDNA infusion only, and pDNA infusion followed by uncontrolled EP at 10 pulses in 10ms duration at an electric field strength of 350V/cm using the same electrode.

The quantification of bioluminescence will be started at 24 hours by injection of D-fluorescein (peritoneal cavity, 15mg/ml of 200. Mu.l). The luminescence of these tumors will be captured with an in vivo imaging system (Lago, spectroscopic instrument) at 24, 48 and 72 hours. Tumor tissue will be collected, bisected longitudinally, with one half frozen in optimal cutting temperature compound (OTC) and one half fixed in formalin for routine histological analysis. Three independent experiments will be performed, with each experimental group consisting of twelve biological replicates. Data will be analyzed using one-way analysis of variance (i.e., kruskal-Wallis, graphPad Prizm).

Routine organization and Immunohistochemistry (IHC) will be performed on the tumor sections to assess necrosis and specific forms of cell death, such as apoptosis, associated with the mCherry expression space. TdT-mediated dUTP point-cut end labeling (TUNEL) and active caspase 3IHC will be performed and the segments will be evaluated to score the extent of apoptosis. Semi-quantitative analysis will be performed using image J scripts as described. H & E stained slides will be used to assess the degree of inflammatory infiltration and necrosis.

The expected result, FCEP, would result in a reduction in the rate of change of expression between the treated tumors. Larger amounts of pDNA transfection are expected to correlate with higher relative decreases in the calculated time constants. In addition, it is expected that more apoptosis and inflammation will be observed as the relative decrease in time constant increases.

Example 7: (expedition)

Target (target 2) is to examine the feedback control system in vivo by performing an intratumoral immunotherapy experiment aimed at tumor regression.

After in vivo characterization, a set of experiments will be performed with the FCEP system to examine tumor regression and durability of expression. For comparison with published studies and to control the rate of change, a homogeneous melanoma model of the contralateral tumor will be used. B16/OVA melanoma cells (1 x 106/injection site) will be implanted subcutaneously in the flank of albino B6 mice (n = 10/group). When the tumor was 75mm3, one tumor per mouse would be injected with polycistronic plastids (50. Mu.l at 1 mg/ml) encoding interleukin-12 (IL-12), luciferase and mCherry. Expression of this plastid allows for immunotherapy and long-term bioluminescence quantification. The tumor will be pulsed with the FCEP system using 350V/cm with the pulse width set at five time constants for each individual pulse. The EP stop criteria for the first cohort would be selected based on the feedback control cohort in target 1, where the maximum expression of pDNA is independent of the observed histological features. The second feedback cohort will be selected from target 1 by selecting the cohort showing significant expression with the least tissue damage. Control animals for this experiment will contain no treatment, pDNA injection only, and pDNA injection followed by conditions optimized for this tumor model. To apply these conditions, a MedPulser with a 6-electrode applicator would be used to apply 6 rotation pulses, each 100 μ s in duration and with an electric field strength of 1,500V/cm.

Data from these experiments will be collected in two different ways. Tumor growth rates of the treated and contralateral tumors will be collected every 48 hours post-treatment with two-dimensional caliper measurements. The treatment will be initiated by infusion of D-fluorescein (200 μ l at 15mg/mL in the abdominal cavity) starting 48 hours after treatment and then every 4 days. Tumor volume and luminescence data will continue to be observed for up to 30 days or until tumor burden exceeds 1,000mm3, at which point the animal will be euthanized according to established IACUC protocols. Three independent experiments will be performed, comprising 12 animals per experimental group. Data will be analyzed using one-way analysis of variance (Kruskal-Wallis).

In addition to monitoring tumor growth rate, tumor specific neoantigen CD8 responses will also be determined by harvesting spleens at the end of the study. The spleen will be mechanically separated and the red blood cells will be lysed by suspension in ACK buffer. Isolation of splenocytes will be purified by cell isolation medium (lympholysin-M, cedarline) prior to staining. The purified cells will then be mixed with a tetramer solution (e.g., SIINFEKL, TS-5001-2C, MBL). CD8 positive T cells will then be determined by flow cytometry analysis (LSR II, BD).

Expected results-expected FCEP device will produce larger IL-12 and IFN-gamma relative to the published EP method. The greater duration of plastid expression, as well as the long-term survival of tumor-bearing animals treated with FCEP, is attributed to the assurance of treatment success. Enhanced survival would serve as an additional metric to evaluate this system, which should be higher than traditional EP treatment groups with an expected long-term survival of approximately 47% based on similar studies, and the control group would likely not respond to treatment.

Potential problems and alternatives-a possible problem is that CD8 positive T cells may be difficult to assess 30 days after treatment. Where this occurs, a separate group of tumor-bearing animals will be treated with the conditions described in this target. Tumors will be excised from the euthanized animals 14 days after treatment.

Example 8: (expedition)

Goal (goal 3) is to validate the feedback control system by performing intratumoral EP in a heterogeneous spontaneous breast cancer model.

After optimization and testing in a homogeneous tumor model, a set of experiments will be performed to confirm FCEP with a heterogeneous model. These experiments will use a transgenic mouse model expressing the polyomavirus intermediate T antigen in the direction of a mouse mammary tumor virus promoter (MMTV-PyVT), which forms spontaneously palpable breast tumors at an age of 8 to 10 weeks. Plastids expressing IL-12, luciferase and mCherry (50. Mu.l at 1 mg/mL) will be delivered into mammary tumors of MMTV-PyVT mice at an age of 10 weeks. Tumors will be treated with 350V/cm pulses using the stopping criteria from target 2, resulting in the longest mean survival. The control group will contain no treatment, pDNA injection only, and pDNA injection followed by 6 pulses of current clinical parameters at 1,500V/cm for 100 μ s. A total of 10 tumors will be treated with each treatment condition, with two of these tumors being treated in each mouse. The experiment will be run a total of three times.

Utilizing each of the proteins encoded by the plastids will allow for the generation of multiple data streams. Luminescence will be quantified by in vivo imaging every 72 hours up to 21 days after infusion of D-fluorescein (peritoneal cavity, 15mg/ml of 200. Mu.l). A population of 5 animals will be euthanized and tumors collected on

days

7, 14 and 21. The collected tumors will be bisected to directly assess IL-12 expression and determine the percentage of transfected cells. A portion of these excised tumors will be clustered and homogenized. IL-12 expression will be quantified directly from these tumors sampled by ELISA assay (Andy Bio Inc.). The other half of the tumor will be isolated (tumor isolation kit, miltenyl Biotec) and run through a flow cytometer (LSR II, BD) using optics specific for mCherry proteins. This will enable the determination of the percentage of transfected cells. Data will be analyzed using one-way analysis of variance.

Expected results-it is expected that FCEP will produce more reproducible transfections of these heterogeneous tumors than current clinical EP protocols. This will be measured directly from the luminescence data and IL-12 expression. In addition, this novel approach would be expected to correlate with the highest percent transfection.

Potential problems and alternatives-a possible problem that may arise during the course of this study is that expression of IL-12 may be difficult to assess in tumors. Where this occurs, an ELISA will be performed to directly measure luciferase levels. In addition, downstream cytokine interferon gamma will be directly evaluated as IL-12 expression replacement.

A time line. Completion of this phase I effort would be performed within a 12 month period. It is expected that target 1 will last for a total of 3 months. Goal 2 will be completed in 5 months. Finally, goal 3 will be completed in 4 months. This timeline is summarized in table 1.

TABLE 1 time schedule of targets (in months)

Claims

1. A system for providing adaptive control to optimize EP pulse parameters during EP of cells and tissues using an electroporation EP apparatus, the system comprising:

a) A measurement device configured to measure dielectric and conductive properties of the cells and tissue, the measurement device comprising:

i) A voltage sensor configured to measure a voltage across the tissue resulting from each of an excitation signal and an EP pulse applied to the tissue; and

ii) a current sensor configured to measure a current across the tissue resulting from each of the excitation signal and the applied EP pulse;

b) An initialization module configured to initialize EP pulse parameters prior to performing initial electroporation in the cell or tissue, the initialized EP pulse parameters including at least an initial pulse width for the initial electroporation and based at least in part on at least one trained model;

c) A generator configured to apply at least one of the excitation signal and the EP pulse to the tissue via a plurality of distal electrodes surrounding a central probe of the EP device, wherein the plurality of distal electrodes are configured to generate an electric field with the central probe, wherein the voltage sensor and the current sensor of the measurement device measure the voltage and current, respectively, across the cells of the tissue in response to the application of the excitation signal;

d) A controller configured to receive signals from the measurement device corresponding to at least one of the excitation signals and the EP pulses in relation to measured sensor data to fit the measured sensor data to at least one trained model and to process the measured sensor data into diagnostics and updated control parameters, wherein the controller comprises:

i) A pre-processing module to receive the signals from the current and voltage measurements related to the measured sensor data and process the measured sensor data to separate desirable data from undesirable data;

ii) a feature extraction module to extract relevant features from the consensus data;

iii) A diagnostic module to apply at least a portion of the relevant features of the desired data to at least one trained diagnostic model; and

iv) a pulse parameter estimation module to estimate at least one of the initialized EP pulse parameter and a subsequent pulse parameter based on results of at least one of the measured sensor data, the diagnostic module, and the feature extraction module; and

e) A memory module to store the desirable data, the undesirable data, the measured sensor data, and the at least one trained model for feature extraction by the controller.

2. The system according to claim 1, wherein the EP device comprises:

a) A central probe defining at least a central lumen and extending from a proximal end to a distal end, at least a portion of the central probe having a helical geometry to create a channel for delivery of a treatment portion to the tissue, the portion of the central probe having at least one jet port positioned along the helical geometry,

wherein the proximal end of the central probe is configured to receive the treatment portion delivered to the central probe, and

Wherein the distal end of the central probe is open to define an opening for delivery of the treatment portion to the tissue and has a shape configured to pierce the tissue;

b) An applicator at least partially housing the central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact the tissue and retract into the applicator; and

c) At least two oppositely charged electroporation electrodes (EPEs) configured to be positioned around the tissue, the EPEs being adapted to extend from a proximal end to a distal end, the distal end having a needle shape configured to pierce the tissue,

wherein the measurement device is coupled to the EPE and the electrode is adapted to be coupled to the generator to receive at least one of the excitation signal and an electrical waveform for the EP pulse.

3. The system according to claim 1, wherein the EP device comprises:

a) A central probe defining at least a central lumen and having a proximal end and a closed distal end, a tip of the distal end having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the central lumen to an exterior of the central probe;

b) At least one access line positioned in the central lumen and slidable within the central probe, the access line having a proximal end positioned in the central probe and a distal end configured to extend to the exterior of the central probe and retract into the central lumen through the exit port, a tip of the distal end of the access line having a shape configured to pierce through the tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which a therapeutic portion is delivered to the tissue,

wherein the treatment portion is delivered from the central lumen into the channel through the exit port;

c) A ramp integrally formed with or coupled to an inner surface of the central probe, the inner surface defining the central lumen, and the ramp configured to contact and guide the lane line to exit the central probe to the exterior of the central probe;

d) An electrical connector electrically connecting the central probe and the access line to the generator;

e) A small aperture connector connected to the central probe for delivery of the treatment portion;

f) A handle at least partially housing the electrical connector and coupled to the central probe and the proximal end of the access line to facilitate a penetration depth of the central probe and the distal end of the access line; and

g) At least two oppositely charged electrodes configured to be positioned around the tissue, the electrodes extending from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to the generator, receive at least one electrical waveform from the generator, and supply the at least one excitation signal and at least one EP pulse to the tissue, wherein the measurement device is coupled to the electrodes.

4. An apparatus for delivering a therapeutic moiety to cells in a treatment region of a tissue, the apparatus comprising:

a) A central probe defining at least a central lumen and extending from a proximal end to a distal end, at least a portion of the central probe having at least one injection port,

wherein the central probe comprises an open proximal end fluidly connecting the central lumen with a lumen of an injector through which a therapeutic agent is delivered to the central probe,

Wherein the central probe comprises an open distal end having a shape configured to pierce the tissue, wherein the open distal end and the at least one ejection port are configured to deliver the treatment portion into the tissue;

wherein a plurality of distal electrodes surround the central probe and are configured to generate an electric field with the central probe;

b) An applicator at least partially housing the central probe, the applicator having a distal end through which the portion of the central probe is configured to extend outside of the applicator to contact the tissue and retract into the applicator.

5. The apparatus of claim 4, further comprising an electroporation system comprising at least two oppositely charged electroporation electrodes configured to be positioned around the zone, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to an electrode power supply, receive at least one electrical waveform from the power supply, and supply a pulsed electric field sufficient for electroporation to the zone.

6. The apparatus of claim 4, the portion of the central probe having a helical geometry to create a channel for delivery of the treatment portion to the tissue, the at least one ejection port being positioned along the helical geometry.

7. An apparatus for delivering a therapeutic moiety to cells in a treatment region of a tissue, the apparatus comprising:

a) A central probe defining at least a first lumen and extending from a proximal end to a distal end, at least a portion of the central probe being formed from or coated with an electrically conductive material,

wherein the proximal end of the central probe is open and fluidly connects the first lumen with a lumen of an injector through which the treatment portion is delivered to the central probe, an

Wherein the distal end of the central probe is open to define an opening for delivery of the treatment portion into the tissue and has a shape configured to pierce the tissue;

b) An applicator housing the central probe, the applicator having a distal end; and

c) A plurality of distal electrodes positioned around the central probe at the distal end of the applicator and configured to generate an electric field with the portion of the central probe,

Wherein the portion of the central probe and at least a portion of the plurality of distal electrodes are extendable outside of the applicator to cause electroporation of the tissue and are retractable from the tissue to the applicator.

8. The apparatus of claim 7, further comprising an electroporation system comprising at least two oppositely charged electroporation electrodes configured to be positioned around the zone, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to an electrode power supply, receive at least one electrical waveform from the power supply, and supply a pulsed electric field sufficient for electroporation to the zone.

9. The apparatus of claim 7, the portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in the tissue and create a channel for delivery of the treatment portion to the tissue, wherein the portion of the central probe is formed of or coated with an electrically conductive material.

10. An apparatus for delivering a therapeutic moiety to a region of target cells of a tissue, the apparatus comprising:

a) A central probe defining at least a first lumen and having a proximal end and a closed distal end, a tip of the distal end having a needle shape configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the first lumen to an exterior of the central probe, wherein a plurality of distal electrodes surround the central probe and are configured to generate an electric field with the central probe;

b) At least one access line positioned in the first lumen and slidable within the central probe, the access line having a proximal end positioned in the central probe and a distal end configured to extend outside of the central probe and retract into the first lumen through the exit port, a tip of the distal end of the access line having a shape configured to pierce through the tissue and define an opening through which at least a portion of the access line enters the tissue to create a fluid channel through which the therapeutic portion is delivered to the tissue,

wherein the treatment portion is delivered from the first lumen into the channel through the exit port;

c) A ramp integrally formed or coupled with the first lumen, the ramp configured to contact and guide the access line to exit the central probe to the exterior of the central probe;

d) An electrical connector electrically connecting the central probe and the access line to a source of electrical power;

e) A small aperture connector connecting the central probe to a syringe for delivery of the treatment portion; and

f) A handle at least partially housing the electrical connector and coupled to proximal ends of the central probe and the access line to facilitate a penetration depth of the distal ends of the central probe and the access line.

11. The device of claim 10, further comprising an electroporation system comprising at least two oppositely charged electrodes configured to be positioned surrounding the region of target cells, the electrodes adapted to extend from a proximal end to a distal end, a tip of the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to the power source, receive an electrical waveform from the power source, and supply a pulsed electric field sufficient for electroporation to the region of target cells.